Recombinant adenovirus vectored FMDV vaccines and uses thereof

ABSTRACT

The present invention encompasses FMDV vaccines or compositions. The invention encompasses recombinant vectors encoding and expressing FMDV antigens, epitopes or immunogens which can be used to protect animals, in particular ovines, bovines, caprines, or swines, against FMDV.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application62/288,540 filed on Jan. 29, 2016.

This invention was made with Government support. The Government hascertain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to compositions for combating Foot andMouth Disease Virus (FMDV) infection in animals. The present disclosureprovides pharmaceutical compositions comprising an FMDV antigen, methodsof vaccination against FMDV, and kits for use with such methods andcompositions.

BACKGROUND OF THE INVENTION

Foot-and-mouth disease (FMD) is one of the most virulent and contagiousdiseases affecting farm animals. This disease is endemic in numerouscountries in the world, especially in Africa, Asia and South America. Inaddition, epidemic outbreaks can occur periodically. The presence ofthis disease in a country may have very severe economic consequencesresulting from loss of productivity, loss of weight and milk productionin infected herds, and from trade embargoes imposed on these countries.The measures taken against this disease consist of strict application ofimport restrictions, hygiene controls and quarantine, slaughtering sickanimals and vaccination programs using vaccines, either as a preventivemeasure at the national or regional level, or periodically when anepidemic outbreak occurs.

FMD is characterized by its short incubation period, its highlycontagious nature, the formation of ulcers in the mouth and on the feetand sometimes, the death of young animals. FMD affects a number ofanimal species, in particular cattle, pigs, sheep and goats. The agentresponsible for this disease is a ribonucleic acid (RNA) virus belongingto the Aphthovirus genus of the Picornaviridae family (Cooper et al.,Intervirology, 1978, 10, 165-180). At present, at least seven types offoot-and-mouth disease virus (FMDV) are known: the European types (A, Oand C), the African types (SAT1, SAT2 and SAT3) and an Asiatic type(Asia 1). Numerous sub-types have also been distinguished (Kleid et al.Science (1981), 214, 1125-1129).

FMDV is a naked icosahedral virus of about 25 nm in diameter, containinga single-stranded RNA molecule consisting of about 8500 nucleotides,with a positive polarity. This RNA molecule comprises a single openreading frame (ORF), encoding a single polyprotein containing, interalia, the capsid precursor also known as protein P1 or P88. Protein P1is myristylated at its amino-terminal end. During the maturationprocess, protein P1 is cleaved by protease 3C into three proteins knownas VP0, VP1 and VP3 (or 1AB, 1D and 1C respectively; Belsham G. J.,Progress in Biophysics and Molecular Biology, 1993, 60, 241-261). In thevirion, protein VP0 is then cleaved into two proteins, VP4 and VP2 (or1A and 1B respectively). The mechanism for the conversion of proteinsVP0 into VP4 and VP2, and for the formation of mature virions is notknown. Proteins VP1, VP2 and VP3 have a molecular weight of about 26,000Da, while protein VP4 is smaller at about 8,000 Da.

The simple combination of the capsid proteins forms the protomer or 5Smolecule, which is the elementary constituent of the FMDV capsid. Thisprotomer is then complexed into a pentamer to form the 12S molecule. Thevirion results from the encapsidation of a genomic RNA molecule byassembly of twelve 12S pentamers, thus constituting the 146S particles.The viral capsid may also be formed without the presence of an RNAmolecule inside it (hereinafter “empty capsid”). The empty capsid isalso designated as particle 70S. The formation of empty capsids mayoccur naturally during viral replication or may be produced artificiallyby chemical treatment.

Some studies have been done on natural empty capsids. In particular,Rowlands et al. (Rowlands et al., J. Gen. Virol., 1975, 26, 227-238)have shown that the virions of A10 foot-and-mouth disease comprisemainly the four proteins VP1, VP2, VP3 and VP4. By comparison, thenatural empty capsids (not obtained by recombination but purified fromcultures of A10 foot-and-mouth virus) essentially contain the uncleavedprotein VP0; identical results with the A-Pando foot-and-mouth virus aredescribed by Rweyemamu (Rweyemamu et al., Archives of Virology, 1979,59, 69-79). The artificial empty capsids, obtained after dialysis in thepresence of Tris-EDTA and after centrifuging, contain no protein VP4.These artificial capsids are slightly immunogenic according to Rowlandset al., and the natural empty capsids are only immunogenic aftertreatment with formaldehyde to stabilize them, while the antibodyresponse induced by the natural empty capsids in the guinea-pig isnevertheless inconstant, as noted by the author. Moreover, Rowlands etal. and Rweyemamu et al. do not agree on the need to stabilize thenatural empty capsids. For Rweyemamu et al., the absence of treatmentwith formaldehyde is not prejudicial to the level of antigenicity of thenatural empty capsids. The immunogenicity is only tested by theinduction of neutralizing antibodies in the guinea-pig.

The expression of the gene coding for the precursor P1 of the capsidproteins by means of a recombinant baculovirus in insect cells iscompared with the expression of the gene coding for P1 associated withthe protease 3C in E. coli (Grubman et al., Vaccine, 1993, 11, 825-829;Lewis et al., J. Virol., 1991, 65, 6572-6580). The co-expression of P1and 3C in E. coli results in the assembling of empty capsids 70S. Theexpression product of these two constructions produces neutralizingantibodies in guinea-pigs and pigs. The titers obtained with theP1/baculovirus construction are low. These same expression productsinduce partial protection in pigs. However, some pigs protected againstthe disease are not protected against the replication of the challengevirus. However, the E. coli expression system does not myristylate theproteins and the protease 3C is toxic to this cell. Lewis et al.conclude that fundamental questions relating to the make-up of the virusand the structure of the capsid needed to obtain maximum protection inthe animal have not been answered. Furthermore, Grubman et al. statethat it would be necessary to stabilize the empty capsids beforeformulating the vaccine; on this point they agree about the problemsencountered with the empty capsids obtained by extraction from viralcultures (see above).

Fusion proteins containing some or all of protein P1 have also beenobtained by the use of viral vectors, namely a herpes virus or vacciniavirus. CA-A-2,047,585 in particular describes a bovine herpes virus usedto produce fusion proteins containing a peptide sequence of thefoot-and-mouth virus (amino acids 141 to 158 of P1 bound to amino acids200 to 213 of P1) fused with the glycoprotein gpIII of this bovineherpes virus. Adenovirus vector has been used to express FMDV emptyvirus capsid (U.S. Pat. No. 8,323,663). Viral vectors have also beenused to express stabilized FMDV empty capsid (U.S. Pat. No. 7,531,182,U.S. Ser. No. 14/863,181). Recently, plants and insect cells have beeninvestigated as a source for the production of FMDV antigens (US2011/0236416, U.S. Ser. No. 14/863,181).

It has been reported that maternally derived antibodies (MDA) are ableto inhibit calves' (under 2 years of age cattle) response to vaccinationagainst FMD (Graves, 1963, Journal of Immunology 91:251-256; Brun etal., 1977, Developments in Biological Standardisation, 25:117-122).

SUMMARY OF THE INVENTION

Compositions or vaccines comprising recombinant viral vectors expressingFMDV polypeptide and fragments and variants thereof are provided. TheFMDV antigens and fragments and variants thereof possess immunogenic andprotective properties. The recombinant viral vectors may be adenovirusvectors expressing FMDV antigens.

The recombinant viral vectors can be formulated into vaccines and/orpharmaceutical compositions. Such vaccines or compositions can be usedto vaccinate an animal and provide protection against homologous andheterologous FMDV strains. The vaccines or compositions formulated withpharmaceutically or veterinarily acceptable carrier, excipient,adjuvant, or vehicle offer thermostability improvements and ability tohandle temperature excursions.

Methods for enhanced protection in conventional animals and maternallyderived antibody-positive (MDA-positive) animals against FMDV infectionsare provided. Kits comprising at least one antigenic polypeptide orfragment or variant thereof and instructions for use are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but notintended to limit the disclosure solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings, in which:

FIG. 1 depicts a table summarizing the DNA and Protein sequences.

FIG. 2 depicts the genes for FMDV A24 strain and the A24 (p1-2AB′) genesused in the chimeric A24-A12 construct.

FIG. 3 depicts the genes for FMDV A12 strain and the A12 (3B′/3C) genesused in the chimeric A24-A12 construct.

FIG. 4 depicts the genetic structural identity assay of recombinantadvenovirus vectored A24-A12 FMDV vaccine by PCR.

FIG. 5 depicts the western blot of recombinant advenovirus vectoredA24-A12 FMDV vaccine.

FIG. 6 depicts the genes for FMDV O1M strain used in the recombinantadvenovirus vectored FMDV O1M vaccine.

FIG. 7 depicts the genes for FMDV Irn strain used in the recombinantadvenovirus vectored FMDV Irn vaccine.

FIG. 8 depicts the genes for FMDV Asia strain used in the recombinantadvenovirus vectored FMDV Asia vaccine.

FIG. 9 depicts the percentage protection by O serotype FMDV vaccine atdifferent doses.

FIG. 10 depicts the serology of O serotype FMDV vaccine at differentdoses.

FIG. 11 depicts the viricidal activity of recombinant advenovirusvectored FMDV+ adjuvants at 25° C.

FIG. 12 depicts the viricidal activity of recombinant advenovirusvectored FMDV+ adjuvants at 4° C.

FIG. 13 depicts the geometric mean of FMDV VN titer.

FIG. 14 depicts the geometic mean of SAV titer.

FIG. 15 depicts the geometric mean of FMDV VN titer.

DETAILED DESCRIPTION

Compositions or vaccines comprising recombinant viral vectors expressingFMDV antigens that elicit an immunogenic response in an animal areprovided. The recombinant viral vectors may be adenovirus vectorsexpressing FMDV antigens. The recombinant viral vectors expressing theantigens may be formulated into vaccines or pharmaceutical compositionsand used to elicit or stimulate a protective response in an animal. Inone embodiment the polypeptide antigen is an FMDV structural protein P1(VP4-VP2-Vp3-VP1), nonstructural protein P2 (2A, 2B, and 2C) ornonstructural protein P3 (3A, 3B, 3C and 3D) or active fragment orvariant thereof.

It is recognized that the antigenic polypeptides of the disclosure maybe full length polypeptides or active fragments or variants thereof. By“active fragments” or “active variants” is intended that the fragmentsor variants retain the antigenic nature of the polypeptide. Thus, thepresent disclosure encompasses any FMDV polypeptide, antigen, epitope orimmunogen that elicits an immunogenic response in an animal. The FMDVpolypeptide, antigen, epitope or immunogen may be any FMDV polypeptide,antigen, epitope or immunogen, such as, but not limited to, a protein,peptide or fragment or variant thereof, that elicits, induces orstimulates a response in an animal, such as an ovine, bovine, caprine orporcine.

The simple combination of the capsid proteins forms the protomer or 5Smolecule, which is the elementary constituent of the FMDV capsid. Thisprotomer is then complexed into a pentamer to form the 12S molecule. Thevirion results from the encapsidation of a genomic RNA molecule byassembly of twelve 12S pentamers, thus constituting the 146S particles.The viral capsid may also be formed without the presence of an RNAmolecule inside it (hereinafter “empty capsid”). The empty capsid isalso designated as particle 70S. The formation of empty capsids mayoccur naturally during viral replication or may be produced artificiallyby chemical treatment.

The present disclosure relates to bovine, ovine, caprine, or swinevaccines or compositions which may comprise an effective amount of arecombinant FMDV antigen or a recombinant viral vector expressing FMDVantigen, and a pharmaceutically or veterinarily acceptable carrier,excipient, adjuvant, or vehicle.

In some embodiments, the vaccines further comprise adjuvants, such asthe oil-in-water (O/W) emulsions described in U.S. Pat. No. 7,371,395.

In still other embodiments, the adjuvants include TS6, TS7, TS8 and TS9emulsions, LR3, LR4 and LR6 emulsions, LF2 emulsion, CARBIGEN™ adjuvant,ENABL® advjuant, polyacrylic acid, aluminum hydroxide or aluminumphosphate, saponin, CpG, water-in-oil emultion, and oil-in-wateremulsion, or combinations thereof.

In some embodiments, the response in the animal is a protective immuneresponse.

By “animal” it is intended mammals, birds, and the like. Animal or hostincludes mammals and human. The animal may be selected from the groupconsisting of equine (e.g., horse), canine (e.g., dogs, wolves, foxes,coyotes, jackals), feline (e.g., lions, tigers, domestic cats, wildcats, other big cats, and other felines including cheetahs and lynx),ovine (e.g., sheep), bovine (e.g., cattle, cow), swine (e.g., pig),caprine (e.g., goat), avian (e.g., chicken, duck, goose, turkey, quail,pheasant, parrot, finches, hawk, crow, ostrich, emu and cassowary),primate (e.g., prosimian, tarsier, monkey, gibbon, ape), and fish. Theterm “animal” also includes an individual animal in all stages ofdevelopment, including embryonic and fetal stages.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a”, “an”, and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicate otherwise.

The antigenic polypeptides of the disclosure are capable of protectingagainst FMDV. That is, they are capable of stimulating an immuneresponse in an animal. By “antigen” or “immunogen” means a substancethat induces a specific immune response in a host animal. The antigenmay comprise a whole organism, killed, attenuated or live; a subunit orportion of an organism; a recombinant vector containing an insert withimmunogenic properties; a piece or fragment of DNA capable of inducingan immune response upon presentation to a host animal; a polypeptide, anepitope, a hapten, or any combination thereof. Alternatively, theimmunogen or antigen may comprise a toxin or antitoxin.

The term “immunogenic protein, polypeptide, or peptide” as used hereinincludes polypeptides that are immunologically active in the sense thatonce administered to the host, it is able to evoke an immune response ofthe humoral and/or cellular type directed against the protein. Inembodiments, the protein fragment has substantially the sameimmunological activity as the total protein. Thus, a protein fragmentaccording to the disclosure can comprises or consists essentially of orconsists of at least one epitope or antigenic determinant. An“immunogenic” protein or polypeptide, as used herein, includes thefull-length sequence of the protein, analogs thereof, or immunogenicfragments thereof. By “immunogenic fragment” is meant a fragment of aprotein which includes one or more epitopes and thus elicits theimmunological response described above. Such fragments can be identifiedusing any number of epitope mapping techniques. For example, linearepitopes may be determined by, e.g., concurrently synthesizing largenumbers of peptides on solid supports, the peptides corresponding toportions of the protein molecule, and reacting the peptides withantibodies while the peptides are still attached to the supports. Suchtechniques are known in the art and described in, e.g., U.S. Pat. No.4,708,871; Geysen et al., 1984, PNAS USA, 81(13): 3998-400; Geysen etal., 1985, PNAS USA, 82(1): 178-82. Similarly, conformational epitopesare readily identified by determining spatial conformation of aminoacids such as by, e.g., x-ray crystallography and 2-dimensional nuclearmagnetic resonance. Methods especially applicable to the proteins of T.parva are described in PCT/US2004/022605.

As discussed the disclosure encompasses active fragments and variants ofthe antigenic polypeptide. Thus, the term “immunogenic protein,polypeptide, or peptide” further contemplates deletions, additions andsubstitutions to the sequence, so long as the polypeptide functions toproduce an immunological response as defined herein. The term“conservative variation” denotes the replacement of an amino acidresidue by another biologically similar residue, or the replacement of anucleotide in a nucleic acid sequence so the encoded amino acid residuedoes not change or is another biologically similar residue. In thisregard, some substitutions will generally be conservative in nature,i.e., those substitutions that take place within a family of aminoacids. For example, amino acids are generally divided into fourfamilies: (1) acidic—aspartate and glutamate; (2) basic—lysine,arginine, histidine; (3) non-polar—alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan; and (4) unchargedpolar—glycine, asparagine, glutamine, cysteine, serine, threonine,tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimesclassified as aromatic amino acids. Examples of conservative variationsinclude the substitution of one hydrophobic residue such as isoleucine,valine, leucine or methionine for another hydrophobic residue, or thesubstitution of one polar residue for another polar residue, such as thesubstitution of arginine for lysine, glutamic acid for aspartic acid, orglutamine for asparagine, and the like; or a similar conservativereplacement of an amino acid with a structurally related amino acid thatwill not have a major effect on the biological activity. Proteins havingsubstantially the same amino acid sequence as the reference molecule butpossessing minor amino acid substitutions that do not substantiallyaffect the immunogenicity of the protein are, therefore, within thedefinition of the reference polypeptide. All of the polypeptidesproduced by these modifications are included herein. The term“conservative variation” also includes the use of a substituted aminoacid in place of an unsubstituted parent amino acid provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide.

The term “epitope” refers to the site on an antigen or hapten to whichspecific B cells and/or T cells respond. The term is also usedinterchangeably with “antigenic determinant” or “antigenic determinantsite”. Antibodies that recognize the same epitope can be identified in asimple immunoassay showing the ability of one antibody to block thebinding of another antibody to a target antigen.

An “immunological response” to a composition or vaccine is thedevelopment in the host of a cellular and/or antibody-mediated immuneresponse to a composition or vaccine of interest. Usually, an“immunological response” includes but is not limited to one or more ofthe following effects: the production of antibodies, B cells, helper Tcells, and/or cytotoxic T cells, directed specifically to an antigen orantigens included in the composition or vaccine of interest. Preferably,the host will display either a therapeutic or protective immunologicalresponse so resistance to new infection will be enhanced and/or theclinical severity of the disease reduced. Such protection will bedemonstrated by either a reduction or lack of symptoms normallydisplayed by an infected host, a quicker recovery time and/or a loweredviral titer in the infected host.

Synthetic antigens are also included within the definition, for example,polyepitopes, flanking epitopes, and other recombinant or syntheticallyderived antigens. Immunogenic fragments for purposes of the disclosurewill usually include at least about 3 amino acids, at least about 5amino acids, at least about 10-15 amino acids, or about 15-25 aminoacids or more amino acids, of the molecule. There is no critical upperlimit to the length of the fragment, which could comprise nearly thefull-length of the protein sequence, or even a fusion protein comprisingat least one epitope of the protein.

Accordingly, a minimum structure of a polynucleotide expressing anepitope is that it comprises or consists essentially of or consists ofnucleotides encoding an epitope or antigenic determinant of an FMDVpolypeptide. A polynucleotide encoding a fragment of an FMDV polypeptidemay comprise or consist essentially of or consist of a minimum of 15nucleotides, about 30-45 nucleotides, about 45-75, or at least 57, 87 or150 consecutive or contiguous nucleotides of the sequence encoding thepolypeptide.

The term “nucleic acid” and “polynucleotide” refers to RNA or DNA thatis linear or branched, single or double stranded, or a hybrid thereof.The term also encompasses RNA/DNA hybrids. The following arenon-limiting examples of polynucleotides: a gene or gene fragment,exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probesand primers. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and nucleotide analogs, uracyl, other sugars andlinking groups such as fluororibose and thiolate, and nucleotidebranches. The sequence of nucleotides may be further modified afterpolymerization, such as by conjugation, with a labeling component. Othertypes of modifications included in this definition are caps,substitution of one or more of the naturally occurring nucleotides withan analog, and introduction of means for attaching the polynucleotide toproteins, metal ions, labeling components, other polynucleotides orsolid support. The polynucleotides can be obtained by chemical synthesisor derived from a microorganism.

The term “gene” is used broadly to refer to any segment ofpolynucleotide associated with a biological function. Thus, genesinclude introns and exons as in genomic sequence, or just the codingsequences as in cDNAs and/or the regulatory sequences required for theirexpression. For example, gene also refers to a nucleic acid fragmentthat expresses mRNA or functional RNA, or encodes a specific protein,and which includes regulatory sequences.

The disclosure further comprises a complementary strand to apolynucleotide encoding an FMDV antigen, epitope or immunogen. Thecomplementary strand can be polymeric and of any length, and can containdeoxyribonucleotides, ribonucleotides, and analogs in any combination.

The terms “protein”, “peptide”, “polypeptide” and “polypeptide fragment”are used interchangeably herein to refer to polymers of amino acidresidues of any length. The polymer can be linear or branched, it maycomprise modified amino acids or amino acid analogs, and it may beinterrupted by chemical moieties other than amino acids. The terms alsoencompass an amino acid polymer that has been modified naturally or byintervention; for example disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling or bioactivecomponent.

An “isolated” biological component (such as a nucleic acid or protein ororganelle) refers to a component that has been substantially separatedor purified away from other biological components in the cell of theorganism in which the component naturally occurs, for instance, otherchromosomal and extra-chromosomal DNA and RNA, proteins, and organelles.Nucleic acids and proteins that have been “isolated” include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids and proteins prepared by recombinanttechnology as well as chemical synthesis.

The term “purified” as used herein does not require absolute purity;rather, it is intended as a relative term. Thus, for example, a purifiedpolypeptide preparation is one in which the polypeptide is more enrichedthan the polypeptide is in its natural environment. In some instancesthe polypeptide is separated from cellular components. By “substantiallypurified” it is intended that so the polypeptide represents severalembodiments at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, or at least 98%, or more of the cellular components ormaterials have been removed. Likewise, the polypeptide may be partiallypurified. By “partially purified” is intended that less than 60% of thecellular components or material is removed. The same applies topolynucleotides. The polypeptides disclosed herein can be purified byany of the means known in the art.

As noted above, the antigenic polypeptides or fragments or variantsthereof are FMDV antigenic polypeptides that are produced by a viralvector in vivo. Fragments and variants of the disclosed polynucleotidesand polypeptides encoded thereby are also encompassed by the presentdisclosure. By “fragment” is intended a portion of the polynucleotide ora portion of the antigenic amino acid sequence encoded thereby.Fragments of a polynucleotide may encode protein fragments that retainthe biological activity of the native protein and hence have immunogenicactivity as noted elsewhere herein. Fragments of the polypeptidesequence retain the ability to induce a protective immune response in ananimal.

“Variants” is intended to mean substantially similar sequences. Forpolynucleotides, a variant comprises a deletion and/or addition of oneor more nucleotides at one or more sites within the nativepolynucleotide and/or a substitution of one or more nucleotides at oneor more sites in the native polynucleotide. As used herein, a “native”polynucleotide or polypeptide comprises a naturally occurring nucleotidesequence or amino acid sequence, respectively. Variants of a particularpolynucleotide of the disclosure (e.g., the reference polynucleotide)can also be evaluated by comparison of the percent sequence identitybetween the polypeptide encoded by a variant polynucleotide and thepolypeptide encoded by the reference polynucleotide. “Variant” proteinis intended to mean a protein derived from the native protein bydeletion or addition of one or more amino acids at one or more sites inthe native protein and/or substitution of one or more amino acids at oneor more sites in the native protein. Variant proteins encompassed by thepresent disclosure are biologically active, that is they the ability toelicit an immune response.

In one aspect, the present disclosure provides FMDV polypeptides fromovine, bovine, caprine, or swine FMDV isolates. In another aspect, thepresent disclosure provides a polypeptide having a sequence as set forthin SEQ ID NO: 2, 4, 6, or 8, and variant or fragment thereof.

Moreover, homologs of FMDV polypeptides from ovine, bovine, caprine, orswine are intended to be within the scope of the present disclosure. Asused herein, the term “homologs” includes orthologs, analogs andparalogs. The term “analogs” refers to two polynucleotides orpolypeptides that have the same or similar function, but that haveevolved separately in unrelated organisms. The term “orthologs” refersto two polynucleotides or polypeptides from different species, but thathave evolved from a common ancestral gene by speciation. Normally,orthologs encode polypeptides having the same or similar functions. Theterm “paralogs” refers to two polynucleotides or polypeptides that arerelated by duplication within a genome. Paralogs usually have differentfunctions, but these functions may be related. Analogs, orthologs, andparalogs of a wild-type FMDV polypeptide can differ from the wild-typeFMDV polypeptide by post-translational modifications, by amino acidsequence differences, or by both. In particular, homologs of thedisclosure will generally exhibit at least 80-85%, 85-90%, 90-95%, or95%, 96%, 97%, 98%, 99% sequence identity, with all or part of thewild-type FMDV polypepetide or polynucleotide sequences, and willexhibit a similar function. Variants include allelic variants. The term“allelic variant” refers to a polynucleotide or a polypeptide containingpolymorphisms that lead to changes in the amino acid sequences of aprotein and that exist within a natural population (e.g., a virusspecies or variety). Such natural allelic variations can typicallyresult in 1-5% variance in a polynucleotide or a polypeptide. Allelicvariants can be identified by sequencing the nucleic acid sequence ofinterest in a number of different species, which can be readily carriedout by using hybridization probes to identify the same gene geneticlocus in those species. Any and all such nucleic acid variations andresulting amino acid polymorphisms or variations that are the result ofnatural allelic variation and that do not alter the functional activityof gene of interest, are intended to be within the scope of thedisclosure.

As used herein, the term “derivative” or “variant” refers to apolypeptide, or a nucleic acid encoding a polypeptide, that has one ormore conservative amino acid variations or other minor modifications so(1) the corresponding polypeptide has substantially equivalent functionwhen compared to the wild type polypeptide or (2) an antibody raisedagainst the polypeptide is immunoreactive with the wild-typepolypeptide. These variants or derivatives include polypeptides havingminor modifications of the FMDV polypeptide primary amino acid sequencesthat may result in peptides which have substantially equivalent activityas compared to the unmodified counterpart polypeptide. Suchmodifications may be deliberate, as by site-directed mutagenesis, or maybe spontaneous. The term “variant” further contemplates deletions,additions and substitutions to the sequence, so long as the polypeptidefunctions to produce an immunological response as defined herein.

The term “conservative variation” denotes the replacement of an aminoacid residue by another biologically similar residue, or the replacementof a nucleotide in a nucleic acid sequence so the encoded amino acidresidue does not change or is another biologically similar residue. Inthis regard, particularly preferred substitutions will generally beconservative in nature, as described above.

The polynucleotides of the disclosure include sequences that aredegenerate as a result of the genetic code, e.g., optimized codon usagefor a specific host. As used herein, “optimized” refers to apolynucleotide that is genetically engineered to increase its expressionin a given species. To provide optimized polynucleotides coding for FMDVpolypeptides, the DNA sequence of the FMDV protein gene can be modifiedto 1) comprise codons preferred by highly expressed genes in aparticular species; 2) comprise an A+T or G+C content in nucleotide basecomposition to that substantially found in said species; 3) form aninitiation sequence of said species; or 4) eliminate sequences thatcause destabilization, inappropriate polyadenylation, degradation andtermination of RNA, or that form secondary structure hairpins or RNAsplice sites. Increased expression of FMDV protein in said species canbe achieved by utilizing the distribution frequency of codon usage ineukaryotes and prokaryotes, or in a particular species. The term“frequency of preferred codon usage” refers to the preference exhibitedby a specific host cell in usage of nucleotide codons to specify a givenamino acid. There are 20 natural amino acids, most of which arespecified by more than one codon. Therefore, all degenerate nucleotidesequences are included in the disclosure as long as the amino acidsequence of the FMDV polypeptide encoded by the nucleotide sequence isfunctionally unchanged.

The sequence identity between two amino acid sequences may beestablished by the NCBI (National Center for Biotechnology Information)pairwise blast and the blosum62 matrix, using the standard parameters(see, e.g., the BLAST or BLASTX algorithm available on the “NationalCenter for Biotechnology Information” (NCBI, Bethesda, Md., USA) server.

The “identity” with respect to sequences can refer to the number ofpositions with identical nucleotides or amino acids divided by thenumber of nucleotides or amino acids in the shorter of the two sequenceswherein alignment of the two sequences can be determined in accordancewith the Wilbur and Lipman algorithm (1983, Proc. Natl. Acad. Sci. USA,vol 80, pp 726-730). The sequence identity or sequence similarity of twoamino acid sequences, or the sequence identity between two nucleotidesequences can be determined using Vector NTI software package(Invitrogen, 1600 Faraday Ave., Carlsbad, Calif.). When RNA sequencesare said to be similar, or have a degree of sequence identity orhomology with DNA sequences, thymidine (T) in the DNA sequence isconsidered equal to uracil (U) in the RNA sequence. Thus, RNA sequencesare within the scope of the disclosure and can be derived from DNAsequences, by thymidine (T) in the DNA sequence being considered equalto uracil (U) in RNA sequences.

Hybridization reactions can be performed under conditions of different“stringency.” See for example, “Molecular Cloning: A Laboratory Manual”,4th edition (Sambrook et al., 2014).

The disclosure further encompasses the FMDV polynucleotides contained ina vector molecule or an expression vector and operably linked to apromoter element and optionally to an enhancer.

A “vector” refers to a recombinant DNA or RNA plasmid or virus thatcomprises a heterologous polynucleotide to be delivered to a targetcell, either in vitro or in vivo. The heterologous polynucleotide maycomprise a sequence of interest for purposes of prevention or therapy,and may optionally be in the form of an expression cassette. As usedherein, a vector needs not be capable of replication in the ultimatetarget cell or subject. The term includes cloning vectors and viralvectors.

The term “recombinant” means a polynucleotide semisynthetic, orsynthetic origin which either does not occur in nature or is linked toanother polynucleotide in an arrangement not found in nature.

“Heterologous” means derived from a genetically distinct entity from therest of the entity to which it is being compared. For example, apolynucleotide may be placed by genetic engineering techniques into aplasmid or vector derived from a different source, and is a heterologouspolynucleotide. A promoter removed from its native coding sequence andoperatively linked to a coding sequence other than the native sequenceis a heterologous promoter.

The present disclosure relates to ovine, bovine, caprine and swinevaccines or pharmaceutical or immunological compositions which maycomprise an effective amount of a recombinant FMDV antigens and apharmaceutically or veterinarily acceptable carrier, adjuvant,excipient, or vehicle.

The subject matter described herein is directed in part, to compositionsand methods related to the FMDV antigen prepared in a baculovirus/insectcell expression system that is highly immunogenic and protects animalsagainst challenge from homologous and heterologous FMDV strains.

Compositions

The present disclosure relates to FMDV vaccines or compositions whichmay comprise an effective amount of a recombinant FMDV antigen and apharmaceutically or veterinarily acceptable carrier, excipient,adjuvant, or vehicle. In one embodiment, the FMDV vaccine or compositioncomprises a recombinant viral vector expressing FMDV antigens.

One embodiment of the disclosure relates to a vaccine or compositioncomprising a viral vector expressing FMDV antigens. The FMDV antigensare obtained by expression of the cDNA of regions P1 (VP4-VP2-VP3-VP1),2A/2B′/3B′ and 3C, or P1 (VP4-VP2-VP3-VP1), 2A/2B/2C and 3A/3B/3C/3D.The structural region P1 and the nonstructural regions P2 or P3 mayderive from the same FMDV serotype or different serotype (chimericantigens).

The present disclosure encompasses any FMDV polypeptide, antigen,epitope or immunogen that elicits an immunogenic response in an animal,such as an ovine, bovine, caprine or swine. The FMDV polypeptide,antigen, epitope or immunogen may be any FMDV polypeptide, antigen,epitope or immunogen, such as, but not limited to, a protein, peptide orfragment thereof, that elicits, induces or stimulates a response in ananimal, such as an ovine, bovine, caprine or swine.

In an embodiment wherein the FMDV immunological composition or vaccineis a recombinant immunological composition or vaccine, the compositionor vaccine comprises a recombinant vector and a pharmaceutical orveterinary acceptable excipient, carrier, adjuvant or vehicle; therecombinant vector is a baculovirus expression vector which may comprisea polynucleotide encoding an FMDV polypeptide, antigen, epitope orimmunogen. The FMDV polypeptide, antigen, epitope or immunogen, may beVP1, VP2, VP3, VP4, 2A, 2B, 2C, 3A, 3B, 3C, or 3D, or any combinationthereof.

In one embodiment, P1 (VP4-VP2-VP3-VP1)-2A/partial 2B/partial 3B and 3Cpolypeptides may be expressed in a viral vector and the expression maybe regulated by one or more promoter sequences. In another embodiment,the FMDV antigen may be chimeric antigen comprising P1(VP4-VP2-VP3-VP1)-2A-partial 2B from FMDV serotype A24 and partial 3Bfrom FMDV serotype A12 and 3C antigen from FMDV serotype A24. In yetanother embodiment, the FMDV antigen may be P1(VP4-VP2-VP3-VP1)-2A-2B-partial 2C-partial 3A-3B-3C.

In another embodiment, the FMDV antigen may be derived from FMDV O1Manisa, O1 BFS or Campos, A24 Cruzeiro, A12, Asia 1 Shamir, A Iran'96,Asia/IRN/05, A22 Iraq, SAT2 Saudi Arabia.

The present disclosure relates to an FMDV vaccine which may comprise aneffective amount of a recombinant FMDV antigen or a recombinant viralvector expressing an FMDV antigen, and a pharmaceutically orveterinarily acceptable carrier, excipient, adjuvant, or vehicle.

In another embodiment, pharmaceutically or veterinarily acceptablecarrier, excipient, adjuvant, or vehicle may be a water-in-oil emulsion.In yet another embodiment, the pharmaceutically or veterinarilyacceptable carrier, excipient, adjuvant, or vehicle may be anoil-in-water emulsion.

The disclosure further encompasses the FMDV polynucleotides contained ina vector molecule or an expression vector and operably linked to apromoter element and optionally to an enhancer.

In one aspect, the present disclosure provides FMDV polypeptides,particularly ovine, bovine, caprine or swine polypeptides having asequence as set forth in SEQ ID NO: 2, 4, 6, or 8, and variants orfragments thereof.

In another aspect, the present disclosure provides a polypeptide havingat least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, 96%, 97%, 98% or 99% sequence identity to an antigenicpolypeptide of the disclosure, particularly to the polypeptides having asequence as set forth in SEQ ID NO: 2, 4, 6, or 8.

In yet another aspect, the present disclosure provides fragments andvariants of the FMDV polypeptides identified above (SEQ ID NO: 2, 4, 6,or 8) which may readily be prepared by one of skill in the art usingmolecular biology techniques.

Variants are homologous polypeptides having an amino acid sequence atleast 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to theamino acid sequence as set forth in SEQ ID NO: 2, 4, 6, or 8.

An immunogenic fragment of an FMDV polypeptide includes at least 8, 10,15, or 20 consecutive amino acids, at least 21 amino acids, at least 23amino acids, at least 25 amino acids, or at least 30 amino acids of anFMDV polypeptide having a sequence as set forth in SEQ ID NO: 2, 4, 6,or 8, or variants thereof. In another embodiment, a fragment of an FMDVpolypeptide includes a specific antigenic epitope found on a full-lengthFMDV polypeptide.

In another aspect, the present disclosure provides a polynucleotideencoding an FMDV polypeptide, such as a polynucleotide encoding apolypeptide having a sequence as set forth in SEQ ID NO: 2, 4, 6, or 8.In yet another aspect, the present disclosure provides a polynucleotideencoding a polypeptide having at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, 96%, 97%, 98% or 99% sequenceidentity to a polypeptide having a sequence as set forth in SEQ ID NO:2, 4, 6, or 8, or a conservative variant, an allelic variant, a homologor an immunogenic fragment comprising at least eight or at least tenconsecutive amino acids of one of these polypeptides, or a combinationof these polypeptides.

In another aspect, the present disclosure provides a polynucleotidehaving a nucleotide sequence as set forth in SEQ ID NO:1, 3, 5, or 7, ora variant thereof. In yet another aspect, the present disclosureprovides a polynucleotide having at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 95%, 96%, 97%,98%, or 99% sequence identity to one of a polynucleotide having asequence as set forth in SEQ ID NO: 1, 3, 5, or 7, or a variant thereof.

The polynucleotides of the disclosure may comprise additional sequences,such as additional encoding sequences within the same transcriptionunit, controlling elements such as promoters, ribosome binding sites,enhancer, 5′UTR, 3′UTR, transcription terminators, polyadenylationsites, additional transcription units under control of the same or adifferent promoter, sequences that permit cloning, expression,homologous recombination, and transformation of a host cell, and anysuch construct as may be desirable to provide embodiments of thisdisclosure.

Elements for the expression of an FMDV polypeptide, antigen, epitope orimmunogen are advantageously present in an inventive vector. In minimummanner, this comprises, consists essentially of, or consists of aninitiation codon (ATG), a stop codon and a promoter, and optionally alsoa polyadenylation sequence for certain vectors such as plasmid andcertain viral vectors, e.g., viral vectors other than poxviruses. Whenthe polynucleotide encodes a polyprotein fragment, e.g., an FMDVpeptide, advantageously, in the vector, an ATG is placed at 5′ of thereading frame and a stop codon is placed at 3′. Other elements forcontrolling expression may be present, such as enhancer sequences,stabilizing sequences, such as intron and signal sequences permittingthe secretion of the protein.

The present disclosure also relates to preparations comprising vectors,such as expression vectors, e.g., therapeutic compositions. Thepreparations can comprise one or more vectors, e.g., expression vectors,such as in vivo expression vectors, comprising and expressing one ormore FMDV polypeptides, antigens, epitopes or immunogens. In oneembodiment, the vector contains and expresses a polynucleotide thatcomprises, consists essentially of, or consists of a polynucleotidecoding for (and advantageously expressing) an FMDV antigen, epitope orimmunogen, in a pharmaceutically or veterinarily acceptable carrier,excipient or vehicle. Thus, according to an embodiment of thedisclosure, the other vector or vectors in the preparation comprises,consists essentially of or consists of a polynucleotide that encodes,and under appropriate circumstances the vector expresses one or moreother proteins of an FMDV polypeptide, antigen, epitope or immunogen, ora fragment thereof.

According to another embodiment, the vector or vectors in thepreparation comprise, or consist essentially of, or consist ofpolynucleotide(s) encoding one or more proteins or fragment(s) thereofof an FMDV polypeptide, antigen, epitope or immunogen, the vector orvectors expressing the polynucleotide(s). In another embodiment, thepreparation comprises one, two, or more vectors comprisingpolynucleotides encoding and expressing, advantageously in vivo, an FMDVpolypeptide, antigen, fusion protein or an epitope thereof. Thedisclosure is also directed at mixtures of vectors that comprisepolynucleotides encoding and expressing different FMDV polypeptides,antigens, epitopes or immunogens, e.g., an FMDV polypeptide, antigen,epitope or immunogen from different animal species such as, but notlimited to, ovine, bovine, caprine or swine.

According to a yet further embodiment of the disclosure, the expressionvector is a plasmid vector or a DNA plasmid vector, in particular an invivo expression vector. In a specific, non-limiting example, the pVR1020or 1012 plasmid (VICAL, Inc.; Luke et al., 1997; Hartikka et al., 1996,Hum Gene Ther, 7(10): 1205-17; see, e.g., U.S. Pat. Nos. 5,846,946 and6,451,769) can be utilized as a vector for the insertion of apolynucleotide sequence. The pVR1020 plasmid is derived from pVR1012 andcontains the human tPA signal sequence. In an embodiment, the human tPAsignal comprises from amino acid M(1) to amino acid S(23) in Genbankunder the accession number HUMTPA14. In another specific, non-limitingexample, the plasmid utilized as a vector for the insertion of apolynucleotide sequence can contain the signal peptide sequence ofequine IGF1 from amino acid M(24) to amino acid A(48) in Genbank underthe accession number U28070. Additional information on DNA plasmidswhich may be consulted or employed in the practice are found, forexample, in U.S. Pat. Nos. 6,852,705; 6,818,628; 6,586,412; 6,576,243;6,558,674; 6,464,984; 6,451,770; 6,376,473; and 6,221,362.

The term plasmid covers any DNA transcription unit comprising apolynucleotide according to the disclosure and the elements necessaryfor its in vivo expression in a cell or cells of the desired host ortarget; and, in this regard, it is noted that a supercoiled ornon-supercoiled, circular plasmid, as well as a linear form, areintended to be within the scope of the disclosure.

A plasmid can comprises or contains or consists essentially of, inaddition to the polynucleotide encoding an FMDV antigen, epitope orimmunogen, optionally fused with a heterologous peptide sequence,variant, analog or fragment, operably linked to a promoter or under thecontrol of a promoter or dependent upon a promoter. In general, it isadvantageous to employ a strong promoter functional in eukaryotic cells.The strong promoter may be, but not limited to, the immediate earlycytomegalovirus promoter (CMV-IE) of human or murine origin, oroptionally having another origin such as the rat or guinea pig, theSuper promoter (Ni, M. et al., Plant J. 7, 661-676, 1995.). The CMV-IEpromoter can comprise the actual promoter part, which may or may not beassociated with the enhancer part. Reference can be made to EP-A-260148, EP-A-323 597, U.S. Pat. Nos. 5,168,062, 5,385,839, and 4,968,615,as well as to PCT Application No WO87/03905. In embodiments, the CMV-IEpromoter is a human CMV-IE (Boshart et al., 1985, Cell, 41(2): 521-30)or murine CMV-IE.

In more general terms, the promoter is of a viral, a plant, or acellular origin. A strong viral promoter other than CMV-IE that may beemployed in the practice of the disclosure is the early/late promoter ofthe SV40 virus or the LTR promoter of the Rous sarcoma virus. A strongcellular promoter that may be usefully employed in the practice of thedisclosure is the promoter of a gene of the cytoskeleton, such as, e.g.,the desmin promoter (Kwissa et al., 2000, Vaccine, 18(22): 2337-44), orthe actin promoter (Miyazaki et al., 1989, Gene, 79(2): 269-77).

The plasmids may comprise other expression control elements. It isparticularly advantageous to incorporate stabilizing sequence(s), e.g.,intron sequence(s), for example, maize alcohol dehydrogenase intron(Callis et al. Genes & Dev. 1(10):1183-1200, December 1987), the firstintron of the hCMV-IE (PCT Application No. WO1989/01036), the intron IIof the rabbit β-globin gene (van Ooyen et al., 1979, Science, 206(4416):337-44). In another embodiment, the plasmids may comprise 3′ UTR. The 3′UTR may be, but not limited to, agrobacterium nopaline synthase (Nos) 3′UTR (Nopaline synthase: transcript mapping and DNA sequence. Depicker,A. et al. J. Mol. Appl. Genet., 1982; Bevan, NAR, 1984, 12(22):8711-8721).

As to the polyadenylation signal (polyA) for the plasmids and viralvectors other than poxviruses, use can more be made of the poly(A)signal of the bovine growth hormone (bGH) gene (see U.S. Pat. No.5,122,458), or the poly(A) signal of the rabbit β-globin gene or thepoly(A) signal of the SV40 virus.

A “host cell” denotes a prokaryotic or eukaryotic cell that has beengenetically altered, or is capable of being genetically altered byadministration of an exogenous polynucleotide, such as a recombinantplasmid or vector. When referring to genetically altered cells, the termrefers both to the originally altered cell and to the progeny thereof.

In one embodiment, the recombinant FMDV antigen is expressed in insectcells.

Methods of Use

In an embodiment, the subject matter disclosed herein is directed to amethod of vaccinating an ovine, bovine, caprine, or swine comprisingadministering to the ovine, bovine, caprine, or swine an effectiveamount of a vaccine which may comprise a recombinant viral vectorexpressing an FMDV antigen, and a pharmaceutically or veterinarilyacceptable carrier, excipient, adjuvant, or vehicle.

In one embodiment of the present disclosure, the method comprises asingle administration of a vaccine composition formulated with anemulsion according to the disclosure. For example, in one embodiment,the immunological or vaccine composition comprises a recombinant viralvector expressing an FMDV antigen.

In another embodiment of the present disclosure, the method comprises asingle administration of two heterologous vaccine compositions. Theheterologous vaccines or compositions may be different types ofvaccines, such as FMDV VLPs vaccine or FMDV viral vector vaccines. Theheterologous vaccines may also be the same type of vaccines expressingthe capsids of different FMDV serotypes, such as A24, A12, O1 Manisa,Asia or Iraq strains.

In an embodiment, the subject matter disclosed herein is directed to amethod of vaccinating an ovine, bovine, caprine, or swine comprisingadministering to the ovine, bovine, caprine, or swine a vaccinecomprising a recombinant viral vector expressing an FMDV antigen invivo.

In an embodiment, the subject matter disclosed herein is directed to amethod of eliciting an immune response comprising administering to theovine, bovine, caprine, or swine a vaccine comprising a recombinantviral vector expressing an FMDV antigen in vivo.

Both homologous and heterologous FMDV strains are used for challenge totest the efficacy of the vaccine. The administering may besubcutaneously or intramuscularly. The administering may be needle free(for example Pigjet or Bioject).

In one embodiment of the disclosure, a prime-boost regimen can beemployed, which is comprised of at least one primary administration andat least one booster administration using at least one commonpolypeptide, antigen, epitope or immunogen. The immunologicalcomposition or vaccine used in primary administration is different innature from those used as a booster. However, it is noted that the samecomposition can be used as the primary administration and the boost.This administration protocol is called “prime-boost”.

A prime-boost according to the present disclosure can include arecombinant viral vector that is used to express an FMDV coding sequenceor fragments thereof encoding an antigenic polypeptide or fragment orvariant thereof. Specifically, the viral vector can express an FMDV geneor fragment thereof that encodes an antigenic polypeptide. Viral vectorcontemplated herein includes, but not limited to, poxvirus [e.g.,vaccinia virus or attenuated vaccinia virus, avipox virus or attenuatedavipox virus (e.g., canarypox, fowlpox, dovepox, pigeonpox, quailpox,ALVAC, TROVAC; see e.g., U.S. Pat. Nos. 5,505,941 and 5,494,807),raccoonpox virus, swinepox virus, etc.], adenovirus (e.g., humanadenovirus, canine adenovirus), herpesvirus (e.g. canine herpesvirus,herpesvirus of turkey, Marek's disease virus, infectiouslaryngotracheitis virus, feline herpesvirus, laryngotracheitis virus(ILTV), bovine herpesvirus, swine herpesvirus), baculovirus, retrovirus,etc. In another embodiment, the avipox expression vector may be acanarypox vector, such as, ALVAC. In yet another embodiment, the avipoxexpression vector may be a fowlpox vector, such as, TROVAC. The FMDVantigen of the disclosure to be expressed is inserted under the controlof a specific poxvirus promoter, e.g., the entomopoxvirus Amsacta moorei42K promoter (Barcena, Lorenzo et al., 2000, J Gen Virol., 81(4):1073-85), the vaccinia promoter 7.5 kDa (Cochran et al., 1985, J Virol,54(1): 30-7), the vaccinia promoter I3L (Riviere et al., 1992, J Virol,66(6): 3424-34), the vaccinia promoter HA (Shida, 1986, Virology,150(2): 451-62), the cowpox promoter ATI (Funahashi et al., 1988, J GenVirol, 69 (1): 35-47), the vaccinia promoter H6 (Taylor et al., 1988,Vaccine, 6(6): 504-8; Guo et al., 1989, J Virol, 63(10): 4189-98; Perkuset al., 1989, J Virol, 63(9): 3829-36.), inter alia.

In another embodiment, the avipox expression vector may be a canarypoxvector, such as, ALVAC. The FMDV antigen, epitope or immunogen may beFMDV P1-3C. The FMDV viral vector may be a canarypox virus such asvCP2186, vCP2181, or vCP2176, or a fowlpox virus such as vFP2215 (seeU.S. Pat. No. 7,527,960). In yet another embodiment, the FMDV antigen,epitope or immunogen may be produced in duckweed (U.S. Published PatentApplication 2011/0236416).

In another aspect of the prime-boost protocol of the disclosure, acomposition comprising the FMDV antigen of the disclosure isadministered followed by the administration of vaccine or compositioncomprising a subunit vaccine comprising FMDV VLPs expressed bybaculovirus in insect cells (see U.S. Ser. No. 14/863,181), or aninactivated viral vaccine or composition comprising the FMDV antigen, ora DNA plasmid vaccine or composition that contains or expresses the FMDVantigen. Likewise, a prime-boost protocol may comprise theadministration of vaccine or composition comprising a subunit vaccinecomprising FMDV VLPs expressed by baculovirus in insect cells, or aninactivated viral vaccine or composition comprising an FMDV antigen, ora DNA plasmid vaccine or composition that contains or expresses an FMDVantigen, followed by the administration of a composition comprising theFMDV antigen of the disclosure. It is further noted that both theprimary and the secondary administrations may comprise the compositioncomprising the FMDV antigen of the disclosure.

A prime-boost protocol comprises at least one prime-administration andat least one boost administration using at least one common polypeptideand/or variants or fragments thereof. The vaccine used inprime-administration may be different in nature from those used as alater booster vaccine. The prime-administration may comprise one or moreadministrations. Similarly, the boost administration may comprise one ormore administrations.

The dose volume of compositions for target species that are mammals,e.g., the dose volume of ovine, bovine, caprine or swine compositions,based on viral vectors, e.g., non-poxvirus-viral-vector-basedcompositions, is generally between about 0.1 to about 5.0 ml, betweenabout 0.1 to about 3.0 ml, and between about 0.5 ml to about 2.5 ml.

The efficacy of the vaccines may be tested about 2 to 4 weeks after thelast immunization by challenging animals, such as ovine, bovine, caprineor swine, with a virulent strain of FMDV, such as the FMDV O1 Manisa, O1BFS or Campos, A24 Cruzeiro, A12, Asia 1 Shamir, A Iran'96, Asia/IRN/05,A22 Iraq, SAT2 Saudi Arabia strains.

Still other strains may include FMDV strains A10-61, A5, A12,A24/Cruzeiro, C3/Indaial, O1, C1-Santa Pau, C1-C5,A22/550/Azerbaijan/65, SAT1-SAT3, A, A/TNC/71/94, A/IND/2/68,A/IND/3/77, A/IND/5/68, A/IND/7/82, A/IND/16/82, A/IND/17/77,A/IND/17/82, A/IND/19/76, A/IND/20/82, A/IND/22/82, A/IND/25/81,A/IND/26/82, A/IND/54/79, A/IND/57/79, A/IND/73/79, A/IND/85/79,A/IND/86/79, A/APA/25/84, A/APN/41/84, A/APS/44/05, A/APS/50/05,A/APS/55/05, A/APS/66/05, A/APS/68/05, A/BIM/46/95, A/GUM/33/84,A/ORS/66/84, A/ORS/75/88, A/TNAn/60/947/Asia/1, A/IRN/05, Asia/IRN/05,O/HK/2001, O/UKG/3952/2001, O/UKG/4141/2001, Asia 1/HNK/CHA/05 (GenBankaccession number EF149010, herein incorporated by reference), Asia I/XJ(Li, ZhiYong et al. Chin Sci Bull, 2007), HK/70 (Chin Sci Bull, 2006,51(17): 2072-2078), O/UKG/7039/2001, O/UKG/9161/2001, O/UKG/7299/2001,O/UKG/4014/2001, O/UKG/4998/2001, O/UKG/9443/2001, O/UKG/5470/2001,O/UKG/5681/2001, O/ES/2001, HKN/2002, O5India, O/BKF/2/92, K/37/84/A,KEN/1/76/A, GAM/51/98/A, A10/Holland, O/KEN/1/91, O/IND49/97,O/IND65/98, O/IND64/98, O/IND48/98, O/IND47/98, O/IND82/97, O/IND81/99,O/IND81/98, O/IND79/97, O/IND78/97, O/IND75/97, O/IND74/97, O/IND70/97,O/IND66/98, O/IND63/97, O/IND61/97, O/IND57/98, O/IND56/98, O/IND55/98,O/IND54/98, O/IND469/98, O/IND465/97, O/IND464/97, O/IND424/97,O/IND423/97, O/IND420/97, O/IND414/97, O/IND411/97, O/IND410/97,O/IND409/97, O/IND407/97, O/IND399/97, O/IND39/97, O/IND391/97,O/IND38/97, O/IND384/97, O/IND380/97, O/IND37/97, O/IND352/97,O/IND33/97, O/IND31/97, O/IND296/97, O/IND23/99, O/IND463/97,O/IND461/97, O/IND427/98, O/IND28/97, O/IND287/99, O/IND285/99,O/IND282/99, O/IND281/97, O/IND27/97, O/IND278/97, O/IND256/99,O/IND249/99, O/IND210/99, O/IND208/99, O/IND207/99, O/IND205/99,O/IND185/99, O/IND175/99, O/IND170/97, O/IND164/99, O/IND160/99,O/IND153/99, O/IND148/99, O/IND146/99, O/SKR/2000, A22/India/17/77.

Further details of these FMDV strains may be found on the EuropeanBioinformatics Information (EMBL-EBI) web pages, and all of theassociated nucleotide sequences are herein incorporated by reference.The inventors contemplate that all FMDV strains, both herein listed, andthose yet to be identified, could be expressed according to theteachings of the present disclosure to produce, for example, effectivevaccine compositions. Both homologous and heterologous strains are usedfor challenge to test the efficacy of the vaccines. The animal may bechallenged intradermally, subcutaneously, spray, intra-nasally,intra-ocularly, intra-tracheally, and/or orally.

The prime-boost administrations may be advantageously carried out 1 to 6weeks apart, for example, about 3 weeks apart. According to oneembodiment, a semi-annual booster or an annual booster, advantageouslyusing the viral vector-based vaccine, is also envisaged. The animals areadvantageously at least 6 to 8 weeks old or about 6 months old at thetime of the first administration.

The compositions comprising the recombinant antigenic polypeptides ofthe disclosure used in the prime-boost protocols are contained in apharmaceutically or veterinary acceptable vehicle, diluent, adjuvant, orexcipient. The protocols of the disclosure protect the animal fromovine, bovine, caprine or porcine FMDV and/or prevent diseaseprogression in an infected animal.

It should be understood by one of skill in the art that the disclosureherein is provided by way of example and the present disclosure is notlimited thereto. From the disclosure herein and the knowledge in theart, the skilled artisan can determine the number of administrations,the administration route, and the doses to be used for each injectionprotocol, without any undue experimentation.

The present disclosure contemplates at least one administration to ananimal of an efficient amount of the therapeutic composition madeaccording to the disclosure. The animal may be male, female, pregnantfemale and newborn. This administration may be via various routesincluding, but not limited to, intramuscular (IM), intradermal (ID) orsubcutaneous (SC) injection or via intranasal or oral administration.The therapeutic composition according to the disclosure can also beadministered by a needleless apparatus (as, for example with a Pigjet,Dermojet, Biojector, Avijet (Merial, Ga., USA), Vetjet or Vitajetapparatus (Bioject, Oregon, USA)). Another approach to administeringplasmid compositions is to use electroporation (see, e.g. Tollefsen etal., 2002; Tollefsen et al., 2003; Babiuk et al., 2002; PCT ApplicationNo. WO99/01158). In another embodiment, the therapeutic composition isdelivered to the animal by gene gun or gold particle bombardment.

In one embodiment, the disclosure provides for the administration of atherapeutically effective amount of a formulation for the delivery andexpression of an FMDV antigen or epitope in a target cell. Determinationof the therapeutically effective amount is routine experimentation forone of ordinary skill in the art. In one embodiment, the formulationcomprises an expression vector comprising a polynucleotide thatexpresses an FMDV antigen or epitope and a pharmaceutically orveterinarily acceptable carrier, vehicle or excipient. In anotherembodiment, the pharmaceutically or veterinarily acceptable carrier,vehicle or excipient facilitates transfection or other means of transferof polynucleotides to a host animal and/or improves preservation of thevector or protein in a host.

In one embodiment, the subject matter disclosed herein provides adetection method for differentiation between infected and vaccinatedanimals (DIVA).

It is disclosed herein that the use of the vaccine or composition of thepresent disclosure allows the detection of FMDV infection in an animal.It is disclosed herein that the use of the vaccine or composition of thepresent disclosure allows the detection of the infection in animals bydifferentiating between infected and vaccinated animals (DIVA). Methodsare disclosed herein for diagnosing the infection of FMDV in an animalusing an FMDV non-structural protein (e.g., a FMDV 3ABC or 3D-specificELISA).

Article of Manufacture

In an embodiment, the subject matter disclosed herein is directed to akit for performing a method of eliciting or inducing an immune responsewhich may comprise any one of the recombinant FMDV immunologicalcompositions or vaccines, or inactivated FMDV immunological compositionsor vaccines, recombinant FMDV viral compositions or vaccines, andinstructions for performing the method.

Another embodiment of the disclosure is a kit for performing a method ofinducing an immunological or protective response against FMDV in ananimal comprising a composition or vaccine comprising an FMDV antigen ofthe disclosure and a recombinant FMDV viral immunological composition orvaccine, and instructions for performing the method of delivery in aneffective amount for eliciting an immune response in the animal.

Another embodiment of the disclosure is a kit for performing a method ofinducing an immunological or protective response against FMDV in ananimal comprising a composition or vaccine comprising an FMDV antigen ofthe disclosure and an inactivated FMDV immunological composition orvaccine, and instructions for performing the method of delivery in aneffective amount for eliciting an immune response in the animal.

Yet another aspect of the present disclosure relates to a kit forprime-boost vaccination according to the present disclosure as describedabove. The kit may comprise at least two vials: a first vial containinga vaccine or composition for the prime-vaccination according to thepresent disclosure, and a second vial containing a vaccine orcomposition for the boost-vaccination according to the presentdisclosure. The kit may contain additional first or second vials foradditional prime-vaccinations or additional boost-vaccinations.

In an embodiment, a composition comprising an FMDV antigen or fragmentor variant thereof and a pharmaceutically or veterinarily acceptablecarrier, excipient, adjuvant, or vehicle is disclosed. In anotherembodiment, a composition comprising a recombinant viral vectorexpressing FMDV antigens and a pharmaceutically or veterinarilyacceptable carrier, excipient, adjuvant, or vehicle is disclosed. Inanother embodiment, the composition described above wherein the FMDVantigen or fragment or variant thereof comprises an immunogenic fragmentcomprising at least 15 amino acids of an ovine, bovine, caprine, orswine FMDV antigen is disclosed. In an embodiment, the abovecompositions wherein the FMDV antigen or fragment or variant thereof ispartially purified are disclosed. In an embodiment, the abovecompositions wherein the FMDV antigen or fragment or variant thereof issubstantially purified are disclosed.

In an embodiment, the above compositions wherein the FMDV antigen orfragment or variant thereof is an ovine, bovine, caprine, or swine FMDVpolypeptide are disclosed. In an embodiment, the above compositionswherein the FMDV polypeptide is a P1 polypeptide, VP0 polypeptide, VP1polypeptide, VP3 polypeptide, VP2 polypeptide, VP4 polypeptide, 2Apolypeptide, 2B polypeptide, 2C polypeptide, 3A polypeptide, 3Bpolypeptide, 3C polypeptide, or 3D polypeptide are disclosed. In anembodiment, the above compositions wherein the FMDV antigen or fragmentor variant thereof has at least 80% sequence identity to the sequence asset forth in SEQ ID NO: 2, 4, 6, or 8 are disclosed. In one embodiment,the above compositions wherein the FMDV antigen is encoded by apolynucleotide having at least 70% sequence identity to the sequence asset forth in SEQ ID NO: 1, 3, 5, or 7 are disclosed. In an embodiment,the above compositions wherein the pharmaceutically or veterinarilyacceptable carrier, excipient, adjuvant, or vehicle is a water-in-oilemulsion or an oil-in-water emulsion are disclosed. In anotherembodiment, a method of vaccinating an animal susceptible to ovine,bovine, caprine, or swine FMDV comprising administering the compositionsabove to the animal is disclosed. In an embodiment, a method ofvaccinating an animal susceptible to ovine, bovine, caprine, or swineFMDV comprising a prime-boost regimen is disclosed. In an embodiment, asubstantially purified antigenic polypeptide expressed in insect cells,wherein the polypeptide comprises: an amino acid sequence having atleast 80% sequence identity to a polypeptide having the sequence as setforth in SEQ ID NO: 2, 4, 6, or 8 is disclosed. In any embodiment theanimal is preferably an ovine, a bovine, a swine, or a caprine. In oneembodiment, a method of diagnosing FMDV infection in an animal isdisclosed. In yet another embodiment, a kit for prime-boost vaccinationcomprising at least two vials, wherein a first vial containing thecomposition comprising an FMDV antigen or fragment or variant thereof,and a second vial containing a recombinant viral vector that contains orexpresses the FMDV antigen is disclosed.

The pharmaceutically or veterinarily acceptable carriers or vehicles oradjuvants or excipients are well known to the one skilled in the art.For example, a pharmaceutically or veterinarily acceptable carrier orvehicle or excipient can be a 0.9% NaCl (e.g., saline) solution or aphosphate buffer. Other pharmaceutically or veterinarily acceptablecarrier or vehicle or excipients that can be used for methods of thisdisclosure include, but are not limited to, poly-(L-glutamate) orpolyvinylpyrrolidone. The pharmaceutically or veterinarily acceptablecarrier or vehicle or adjuvants or excipients may be any compound orcombination of compounds facilitating the administration of the vector(or protein expressed from an inventive vector in vitro);advantageously, the carrier, vehicle or excipient may facilitatetransfection and/or improve preservation of the vector (or protein).Doses and dose volumes are herein discussed in the general descriptionand can also be determined by the skilled artisan from this disclosureread in conjunction with the knowledge in the art, without any undueexperimentation.

The cationic lipids containing a quaternary ammonium salt which areadvantageously but not exclusively suitable for plasmids, areadvantageously those having the following formula:

in which R1 is a saturated or unsaturated straight-chain aliphaticradical having 12 to 18 carbon atoms, R2 is another aliphatic radicalcontaining 2 or 3 carbon atoms and X is an amine or hydroxyl group, e.g.the DMRIE. In another embodiment the cationic lipid can be associatedwith a neutral lipid, e.g. the DOPE.

Among these cationic lipids, preference is given to DMRIE(N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propaneammonium; WO96/34109), advantageously associated with a neutral lipid,advantageously DOPE (dioleoyl-phosphatidyl-ethanol amine; Behr, 1994),to form DMRIE-DOPE.

Advantageously, the plasmid mixture with the adjuvant is formedextemporaneously and advantageously contemporaneously withadministration of the preparation or shortly before administration ofthe preparation; for instance, shortly before or prior toadministration, the plasmid-adjuvant mixture is formed, advantageouslyso as to give enough time prior to administration for the mixture toform a complex, e.g. between about 10 and about 60 minutes prior toadministration, such as approximately 30 minutes prior toadministration.

When DOPE is present, the DMRIE:DOPE molar ratio is about 95:about 5 toabout 5:about 95, more advantageously about 1:about 1, e.g., 1:1.

The DMRIE or DMRIE-DOPE adjuvant:plasmid weight ratio can be betweenabout 50:about 1 and about 1:about 10, such as about 10:about 1 andabout 1:about 5, and about 1:about 1 and about 1:about 2, e.g., 1:1 and1:2.

In another embodiment, pharmaceutically or veterinarily acceptablecarrier, excipient, adjuvant, or vehicle may be a water-in-oil emulsion.Examples of suitable water-in-oil emulsions include oil-basedwater-in-oil vaccinal emulsions which are stable and fluid at 4° C.containing: from 6 to 50 v/v % of an antigen-containing aqueous phase,preferably from 12 to 25 v/v %, from 50 to 94 v/v % of an oil phasecontaining in total or in part a non-metabolizable oil (e.g., mineraloil such as paraffin oil) and/or metabolizable oil (e.g., vegetable oil,or fatty acid, polyol or alcohol esters), from 0.2 to 20 p/v % ofsurfactants, preferably from 3 to 8 p/v %, the latter being in total orin part, or in a mixture either polyglycerol esters, said polyglycerolesters being preferably polyglycerol (poly)ricinoleates, orpolyoxyethylene ricin oils or else hydrogenated polyoxyethylene ricinoils. Examples of surfactants that may be used in a water-in-oilemulsion include ethoxylated sorbitan esters (e.g., polyoxyethylene (20)sorbitan monooleate (TWEEN 80®), available from AppliChem, Inc.,Cheshire, Conn.) and sorbitan esters (e.g., sorbitan monooleate (SPAN80®), available from Sigma Aldrich, St. Louis, Mo.). In addition, withrespect to a water-in-oil emulsion, see also U.S. Pat. No. 6,919,084,e.g., Example 8 thereof, incorporated herein by reference. In someembodiments, the antigen-containing aqueous phase comprises a salinesolution comprising one or more buffering agents. An example of asuitable buffering solution is phosphate buffered saline. In anadvantageous embodiment, the water-in-oil emulsion may be awater/oil/water (W/O/W) triple emulsion (U.S. Pat. No. 6,358,500).Examples of other suitable emulsions are described in U.S. Pat. No.7,371,395.

The immunological compositions and vaccines according to the disclosuremay comprise or consist essentially of one or more adjuvants. Suitableadjuvants for use in the practice of the present disclosure are (1)polymers of acrylic or methacrylic acid, maleic anhydride and alkenylderivative polymers, (2) immunostimulating sequences (ISS), such asoligodeoxyribonucleotide sequences having one or more non-methylated CpGunits (Klinman et al., 1996, PNAS USA, 93(7): 2879-83; WO98/16247), (3)an oil in water emulsion, such as the SPT emulsion described on page 147of “Vaccine Design, The Subunit and Adjuvant Approach” published by M.Powell, M. Newman, Plenum Press 1995, 6: 147, 183, and the emulsion MF59described on page 183 of the same work, (4) cation lipids containing aquaternary ammonium salt, e.g., DDA (5) cytokines, (6) aluminumhydroxide or aluminum phosphate, (7) saponin or (8) other adjuvantsdiscussed in any document cited and incorporated by reference into theinstant application, or (9) any combinations or mixtures thereof.

The oil in water emulsion (3), which is especially appropriate for viralvectors, can be based on: light liquid paraffin oil (Europeanpharmacopoeia type), isoprenoid oil such as squalane, squalene, oilresulting from the oligomerization of alkenes, e.g. isobutene or decene,esters of acids or alcohols having a straight-chain alkyl group, such asvegetable oils, ethyl oleate, propylene glycol, di(caprylate/caprate),glycerol tri(caprylate/caprate) and propylene glycol dioleate, or estersof branched, fatty alcohols or acids, especially isostearic acid esters.

The oil is used in combination with emulsifiers to form an emulsion. Theemulsifiers may be nonionic surfactants, such as: esters of on the onehand sorbitan, mannide (e.g. anhydromannitol oleate), glycerol,polyglycerol or propylene glycol and on the other hand oleic,isostearic, ricinoleic or hydroxystearic acids, said esters beingoptionally ethoxylated, or polyoxypropylene-polyoxyethylene copolymerblocks, such as Pluronic, e.g., L121.

Among the type (1) adjuvant polymers, preference is given to polymers ofcrosslinked acrylic or methacrylic acid, especially crosslinked bypolyalkenyl ethers of sugars or polyalcohols. These compounds are knownunder the name carbomer (Pharmeuropa, vol. 8, no. 2, June 1996). Oneskilled in the art can also refer to U.S. Pat. No. 2,909,462, whichprovides such acrylic polymers crosslinked by a polyhydroxyl compoundhaving at least three hydroxyl groups, preferably no more than eightsuch groups, the hydrogen atoms of at least three hydroxyl groups beingreplaced by unsaturated, aliphatic radicals having at least two carbonatoms. The preferred radicals are those containing 2 to 4 carbon atoms,e.g. vinyls, allyls and other ethylenically unsaturated groups. Theunsaturated radicals can also contain other substituents, such asmethyl. Products sold under the name CARBOPOL® (BF Goodrich, Ohio, USA)are especially suitable. They are crosslinked by allyl saccharose or byallyl pentaerythritol. Among them, reference is made to CARBOPOL® 1974P,934P and 971P.

As to the maleic anhydride-alkenyl derivative copolymers, preference isgiven to EMA® (Monsanto), which are straight-chain or crosslinkedethylene-maleic anhydride copolymers and they are, for example,crosslinked by divinyl ether.

With regard to structure, the acrylic or methacrylic acid polymers andEMA® are preferably formed by basic units having the following formula:

in which:

-   -   R1 and R2, which can be the same or different, represent H or        CH3    -   x=0 or 1, preferably x=1    -   y=1 or 2, with x+y=2.

For EMA®, x=0 and y=2 and for carbomers x=y=1.

These polymers are soluble in water or physiological salt solution (20g/l NaCl) and the pH can be adjusted to 7.3 to 7.4, e.g., by soda(NaOH), to provide the adjuvant solution in which the expressionvector(s) can be incorporated. The polymer concentration in the finalimmunological or vaccine composition can range between about 0.01 toabout 1.5% w/v, about 0.05 to about 1% w/v, and about 0.1 to about 0.4%w/v.

The cytokine or cytokines (5) can be in protein form in theimmunological or vaccine composition, or can be co-expressed in the hostwith the immunogen or immunogens or epitope(s) thereof. Preference isgiven to the co-expression of the cytokine or cytokines, either by thesame vector as that expressing the immunogen or immunogens or epitope(s)thereof, or by a separate vector thereof.

The disclosure comprehends preparing such combination compositions; forinstance by admixing the active components, advantageously together andwith an adjuvant, carrier, cytokine, and/or diluent.

Cytokines that may be used in the present disclosure include, but arenot limited to, granulocyte colony stimulating factor (G-CSF),granulocyte/macrophage colony stimulating factor (GM-CSF), interferon α(IFNα), interferon β (IFNβ), interferon γ, (IFNγ), interleukin-1α(IL-1α), interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-3(IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6(IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9(IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12(IL-12), tumor necrosis factor α (TNFα), tumor necrosis factor β (TNFβ),polyinosinic and polycytidylic acid, cytidine-phosphate-guanosineoligodeoxynucleotides (CpG ODN), and transforming growth factor β(TGFβ). It is understood that cytokines can be co-administered and/orsequentially administered with the immunological or vaccine compositionof the present disclosure. Thus, for instance, the vaccine of theinstant disclosure can also contain an exogenous nucleic acid moleculethat expresses in vivo a suitable cytokine, e.g., a cytokine matched tothis host to be vaccinated or in which an immunological response is tobe elicited (for instance, a bovine cytokine for preparations to beadministered to bovines).

In a particular embodiment, the adjuvant may include TS6, TS7, TS8 andTS9 emulsions (U.S. Pat. No. 7,371,395); LR3 and LR4 (U.S. Pat. No.7,691,368); TSAP (U.S. Published Patent Application 20110129494);TRIGEN™ (Newport Labs); synthetic dsRNAs (e.g. poly-IC, poly-ICLC[HILTONOL®]); CARBIGEN™ adjuvant (MVP Laboratories, Inc.); ENABL®advjuant (VaxLiant); and MONTANIDE™ adjuvants (W/O, W/O/W, O/W, IMS andGel) (SEPPIC). The adjuvant concentration in the final immunologicalcomposition or vaccine composition can range between 5% to 80% v/v.

In the case of immunological composition and/or vaccine based on abaculovirus/insect cell-expressed polypeptides, a dose may include,about 1 μg to about 2000 μg, about 50 μg to about 1000 μg, and fromabout 100 μg to about 500 μg of FMDV antigen, epitope or immunogen. Thedose may include about 10² to about 10²⁰, about 10³ to about 10¹⁸, about10⁴ to about 10¹⁶, about 10⁵ to about 10¹² VLPs (viral like particles).In the case of immunological composition and/or vaccine based on a viralvector expressing FMDV antigens, a dose may include, about 10³ viralparticles to about 10¹⁵ viral particles, about 10³ viral particles toabout 10¹⁴ viral particles, about 10³ viral particles to about 10¹³viral particles, about 10³ viral particles to about 10¹² viralparticles. The viral particles may be calculated based on any virustitration methods including, but not limited to, FFA (Focus FormingAssay) or FFU (Focus Forming Unit), TCID₅₀ (50% Tissue Culture InfectiveDose), PFU (Plaque Forming Units), and FAID₅₀ (50% Fluorescent AntibodyInfectious Dose). The dose volumes can be between about 0.1 and about 10ml, between about 0.2 and about 5 ml.

The disclosure will now be further described by way of the followingnon-limiting examples.

EXAMPLES

Construction of DNA inserts, plasmids and recombinant viral vectors wascarried out using the standard molecular biology techniques described byJ. Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4th edition,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2014).

Example 1 Construction of Recombinant Human Adenovirus 5 Vectored FMDVAntigens Example 1.1 Construction of Recombinant Human Adenovirus 5Vectored FMDV Chimeric A24-A12 Antigen

The Human Adenovirus C, serotype 5 (Ad5) vector (adenovirus vector) withdeletions in the E1, E3, and E4 regions of the genome (GV11 backbone,see U.S. Pat. No. 8,323,663) was used to construct the recombinant FMDVvaccine expressing chimeric FMDV protein. The chimeric FMDV proteincontains structural capsid gene of FMDV serotype A24 (A24 Pl) andnonstructural genes A24 (2A-partial 2B), nonstructural partial 3B geneand the protease gene of FMDV serotype A12 (partial 3B-C3) (SEQ IDNO:2).

The chimeric polynucleotide encoding FMDV A24 (P1-2A-partial 2B)-A12(partial 3B-3C) was introduced into the shuttle plasmid (see FIGS. 2 and3). After co-transformation of the linearized GV11 backbone plasmid(which carries a kanamycin resistance gene) and the linearized shuttleplasmid containing the chimeric FMDV polynucleotide into BJDE3 E. colicells, kanamycin-resistant clones of E. coli were selected. Clonesbearing recombinant plasmids were confirmed by RFLP. A single clone wasselected, the plasmid linearized and transfected into M2A cells. Thelysate from the M2Acell transfection was serially passaged to producehigh-titered stock of recombinant adenovirus vectored chimeric FMDVvaccine.

The donor gene insertion site is between the CMV promoter and SV40 PolyAof an Ad5 E1 expression cassette, and the expression cassette isinserted into the BBA at the E1 region The A24-A12 expression cassette,located at the E 1 region deletion junction, reads right-to-left withrespect to the viral genome RNA transcription. There are no knownregulatory signals in the flanking nucleotide sequence of the insertioncassette. The CMV enhancer/promoter controls the initiation oftranscription. Within this sequence are the viral enhancer CAAT box,TATA box, transcription start site, and 5′ splice site sequences.

The CMV sequences are followed by an artificial untranslated region(UTR) containing a splice donor sequence. The open reading frame of thegene to be expressed follows the 3′ splice site sequences and the SimianVirus 40 (SV40) early polyadenylation signal is positioned 3′ of theopen reading frame to terminate transcription.

Primer pairs were designed for the RBA Genetic Structural Identity (GSI)assay. The GSI assay uses PCR to identify the backbone biological agentand the expression cassette. The GSI blot (see FIG. 4) showed thecorrect backbone and A24-A12 FMDV expression cassette. Sequence analysisof the A24-A12 expression cassette also confirmed the nucleotidesequence identity between the shuttle plasmid and the DNA extracted fromthe recombinant adenovirus vectored chimeric A24-A12 FMDV vaccine.

The recombinant adenovirus vectored chimeric A24-A12 FMDV vaccine wasconfirmed to express A24 protein in vitro in 293 cells by a western blotassay, yielding a reactive protein that migrated to the position ofapproximately 28 KDa (see FIG. 5). The antibody used for detection inthe Western Blot assay is a monoclonal antibody. The molecular weight ofthe A24 protein expressed from the recombinant adenovirus vectoredchimeric A24-A12 FMDV vaccine was indistinguishable from that observedfollowing transfection of the positive control shuttle plasmid into 293cells (see FIG. 5).

Example 1.2 Construction of Recombinant Human Adenovirus 5 Vectored FMDVO1M Antigen

The Human Adenovirus C, serotype 5 (Ad5) vector (adenovirus vector) withdeletions in the E1, E3, and E4 regions of the genome (GV11 backbone,see U.S. Pat. No. 8,323,663) was used to construct the recombinant FMDVvaccine expressing FMDV protein. The FMDV protein contains structuralcapsid P1 (VP4-VP2-VP3-VP1) and the nonstructural proteins 2ABC and 3ABCincluding the full-length protease 3C (SEQ ID NO: 4) from FMDV O1/Man/87strain.

The synthetic polynucleotide (SEQ ID NO: 3) encoding FMDV O1M P1(VP4-VP2-VP3-VP1-2ABC′3A′BC) was introduced into the shuttle plasmid(see FIG. 6). The recombinant adenovirus vectored O1M FMDV vaccine wasconstructed following the procedure as described in Example 1.2.

The O1M87 FMDV expression cassette, located at the E1 region deletionjunction, reads right-to left with respect to the viral genome RNAtranscription. There are no known regulatory signals in the flankingnucleotide sequence of the insertion cassette. The CMV enhancer/promotercontrols the initiation of transcription. Within this sequence are theviral enhancer CAAT box, TATA box, transcription start site, and 5′splice site sequences.

The CMV sequences are followed by an artificial untranslated region(UTR) containing a splice donor sequence. The open reading frame of thegene to be expressed follows the 3′ splice site sequences and the SimianVirus 40 (SV40) early polyadenylation signal is positioned 3′ of theopen reading frame to terminate transcription.

The GSI blot showed the correct backbone and O1M87 FMDV expressioncassette. Sequence analysis of the O1M87 FMDV expression cassette alsoconfirmed the nucleotide sequence identity between the shuttle plasmidand the DNA extracted from the recombinant adenovirus vectored O1M87FMDV vaccine. The recombinant adenovirus vectored O1M87 FMDV vaccine wasconfirmed to express O1M87 protein in vitro in 293 cells by a westernblot assay.

Example 1.3 Construction of Recombinant Human Adenovirus 5 Vectored FMDVIrn Antigen

The Human Adenovirus C, serotype 5 (Ad5) vector (adenovirus vector) withdeletions in the E1, E3, and E4 regions of the genome (GV11 backbone,see U.S. Pat. No. 8,323,663) was used to construct the recombinant FMDVvaccine expressing FMDV protein. The synthetic FMDV capsid codingsequence of P1 and nonstructural genes 2A, 2B, partial 2C (2C′), partial3A (3A′), 3B and the protease coding sequence of 3C of FMDV serotypeA/Irn/05 was introduced into a shuttle plasmid (see FIG. 7). Therecombinant adenovirus vectored Irn FMDV vaccine was constructedfollowing the procedure as described in Example 1.2.

The Irn FMDV expression cassette, located at the E1 region deletionjunction, reads right-to left with respect to the viral genome RNAtranscription. There are no known regulatory signals in the flankingnucleotide sequence of the insertion cassette. The CMV enhancer/promotercontrols the initiation of transcription. Within this sequence is theviral enhancer CAAT box, TATA box, transcription start site, and 5′splice site sequences.

The CMV sequences are followed by an artificial untranslated region(UTR) containing a splice donor sequence. The open reading frame of thegene to be expressed follows the 3′ splice site sequences and the SimianVirus 40 (SV40) early polyadenylation signal is positioned 3′ of theopen reading frame to terminate transcription.

The recombinant adenovirus vectored Irn FMDV vaccine was identifiedusing a PCR-based Genetic Structural Identity (GSI) assay and confirmedusing protein expression by western blot technique.

Example 1.4 Construction of Recombinant Human Adenovirus 5 Vectored FMDVAsia Antigen

The Human Adenovirus C, serotype 5 (Ad5) vector (adenovirus vector) withdeletions in the E1, E3, and E4 regions of the genome (GV11 backbone,see U.S. Pat. No. 8,323,663) was used to construct the recombinant FMDVvaccine expressing FMDV protein. The synthetic FMDV capsid codingsequence of P1 and nonstructural genes 2A, 2B, partial 2C (2C′), partial3A (3A′), 3B and the protease coding sequence of 3C from FMDV strainAsia/Leb/89 was introduced into a shuttle plasmid (see FIG. 8). Therecombinant adenovirus vectored Asia FMDV vaccine was constructedfollowing the procedure as described in Example 1.2.

The Asia FMDV expression cassette, located at the E1 region deletionjunction, reads right-to left with respect to the viral genome RNAtranscription. There are no known regulatory signals in the flankingnucleotide sequence of the insertion cassette. The CMV enhancer/promotercontrols the initiation of transcription. Within this sequence are theviral enhancer CAAT box, TATA box, transcription start site, and 5′splice site sequences.

The CMV sequences are followed by an artificial untranslated region(UTR) containing a splice donor sequence. The open reading frame of thegene to be expressed follows the 3′ splice site sequences and the SimianVirus 40 (SV40) early polyadenylation signal is positioned 3′ of theopen reading frame to terminate transcription.

The recombinant adenovirus vectored Asia FMDV vaccine was confirmed toexpress Asia protein in vitro in 293 cells by a western blot assay,yielding a reactive protein that migrated to the position ofapproximately 38 kDa. The antibody used for detection in the WesternBlot assay is the FMD VP2 polyclonal. The molecular weight of theAsiaSS.2B protein expressed was indistinguishable from that observedfollowing transfection of the positive control plasmid into 293 cells.

Example 2 Challenge Study in Cattles and Pigs

Cattles and pigs were vaccinated with the A24-A12 FMDV vaccine or O1M87FMDV vaccine once at Day 0 via IM and challenged at day 14 by many FMDVserotypes, such as A24, A12, O1, Asia, Irn, and Iraq strains.

FIG. 9 shows the protection of O1 FMDV vaccine in animals against FMDVchallenge at three different doses and control. In this dose titrationstudy, the recombinant adeno-vectored O1M FMDV vaccine was evaluated forthe ability to confer protection against FMD generalized disease (pedallesions) following direct, IDL homologous challenge at 14 dayspost-vaccination (dpv). Healthy 6 month old female Holstein cattle wererandomized to one of four treatment groups. Control, naïve cattle (T01;n=4) were immunized intramuscularly with a single, 2 mL dose of finalformulation buffer (FFB). Cattle in T02-T04 (n=7/group) were vaccinatedwith decreasing doses [Anti-adenovirus hexon focus forming units (FFU)or focus forming assay (FFA); log₁₀] of active ingredient prepared frommaster seed virus passage 2 (MSV+2) formulated in ENABL™ C1 adjuvant.T02-T04 received 2.38×10⁵, 5.94×10⁴ FFU or 1.49×10⁴ FFU total dose,respectively. At 2 weeks post-vaccination (day of challenge), 100% ofT02 and T03 vaccinates had FMDV O1 Manisa serum virus neutralizing (SVN)titers, versus 43% in the lowest vaccine dose treatment group (T04).Following intradermal lingual challenge with 1×10⁴ bovine infectiousdose units 50% (BID₅₀) of FMDV O1 Manisa, 100% of T01 control, naivecattle exhibited generalized disease (pedal lesions). In contrast, thelevel of protection against generalized disease in vaccinated groupsranged from 86% (T04) to 100% (T02 and T03). All four control cattle(T01) were FMDV positive in plasma collected on 1-3 days post-challenge(dpc), whereas none of the twenty-one vaccinates had detectable plasmaviremia on 1-5 dpc. In T01, 88% of the nasal samples collected 2-5 dpcwere virus positive, compared to 25% (T03), 27% (T02) and 36% (T04) ofthe 2-5 dpc tested samples. FIG. 10 shows the serology of O1 FMDVvaccine at three doses and control.

These results demonstrate that the a recombinant adenovector O1M FMDVvaccine active ingredient formulated in ENABL C1 adjuvant is highlyimmunogenic and efficacious against IDL, homologous FMDV challenge incattle, and provides data on the estimated minimum protective dose.

Both A24-A12 and O1M87 vaccines were tested to be safe in calves andmice.

Example 3 Adjuvant Serology Immunogenicity and CorrespondingViricidal/Stability Study

Adjuvanting a vaccine can reduce the minimum protective dose (MPD) andresult in a more effective vaccine. A reduced MPD may be balanced by theadjuvant cost and supply security. However, some adjuvants can reducethe vaccine effectiveness. The adjuvant may push the immune systemtowards an undesirable response or the adjuvant may be detrimental tothe immunizing agent (lack of stability for example).

The objective of the study was to assess the adjuvant serology efficacy.An efficacy serology study was conducted in cattle, and in parallel avirucidal/stability study was performed to determine if any of the fiveadjuvants have detrimental effects on the adenovirus FMDV vaccines. Eachdose consists of 200 μl of final AI (Active Ingredient) per dose at a 2ml dose with each of the adjuvants. The adjuvants are polyacrylic acid,LF2 emulsion, LR6 emulsion, CARBIGEN™ M and ENABL® C1 (see Table 1.1below).

TABLE 1.1 Preparation of adenovirus FMDV vaccines Group Adjuvant Vaccineformulation G1 No adjuvant FMDV vaccine (10^(5.7) per ml) + FFB (finalformulation buffer) (50% v/v) G2 Polyacrylic acid FMDV vaccine (10^(5.7)per ml) + polyacrylic acid polymer (4 mg/2 ml dose) G3 LF2 emulsion FMDVvaccine (10^(5.7) per ml) + TS6* at 20% v/v of final serial G4 LR6emulsion FMDV vaccine (10^(5.7) per ml) + LR4** at 25% v/v final serialG5 CARBIGEN ™ M FMDV vaccine (10^(5.7) per ml) + CARBIGEN ™ M to 10% v/vfinal serial G6 ENABL ® C1 FMDV vaccine (10^(5.7) per ml) + ENABL ® C1(20% in final serial) TS6*: TS6 adjuvant/emulsion as described in U.S.Pat. No. 7,608,279 and U.S. Pat. No. 7,371,395 LR4**: LR4adjuvant/emulsion as described in U.S. Pat. No. 7,691,368 CARBIGEN ™: acarbomer-based (Carbopol 934P) adjuvant suspension, product of MVPLaboratories, Inc. ENABL ® C1: adjuvant product for cattle purchasedcommercially fromVaxLiant

TABLE 1.2 TS6 emulsion (premulsion described in U.S. Pat. No. 7,608,279and U.S. Pat. No. 7,371,395) Oily phase (120 ml) Sorbitan monooleate(SPAN 80 ®) 1.8% w/v Sorbitan trioleate (20 OE) (TWEEN 85 ®) 10.2% w/vParaffin oil (MARCOL 82 ®) 88% v/v Aqueous phase (120 ml) 20% (w/v)solution of sorbitan monooleate (20 OE) 11.25% w/v (TWEEN 80 ®)Phosphate disodic and monopotassic 0.02M isotonic 85.75% v/v buffer (pH7.8) Sodium mercurothiolate (Thionersal ®) 1% in water 1.5% v/v

TABLE 1.3 LR4 emulsion (premulsion described in U.S. Pat. No. 7,691,368)Oily phase (72 ml) Oleth-2 (BRIJ ® 92) 1.8% w/v Oleth-5 (VOLPO ® N5)8.2% w/v Paraffin oil (MARCOL 82 ®) 87.5% v/v Preservative 2.5% v/vAqueous phase (108 ml) Poloxamer 407 (LUTROL ® F127) 0.58% w/v Isotonicbuffer containing disodium and Q.S. to 100.0% v/v monopotassiumphosphate 0.02M (pH 7.8)

Vaccines were stored at 4° C. and 25° C. and tested over time. Theviruses in each vaccine were titrated in accordance with the standardfocus forming assay titration assay wherein the amount of virus detectedis read by specific anti-adenovirus antibodies on the cell monolayer.The respective titers were compared. Data from the first 3 months ispresented below, in Table 2 and in FIGS. 11 (25° C.) and 12 (4° C.). At4° C., most antigens or viruses present in the formulated vaccines arestable out to 3 months. At 25° C., antigens or viruses formulated withadjuvants polyacrylic acid, LF2 and Carbigen M are relatively stable outto 7 weeks. The stability of these three formulations showed a slightdecrease at 25° C. at 3-months point. However, this drop is less whenthe recombinant viruse is combined with adjuvant confirming theprotective effect of adjuvant. The antigens or viruses formulated withadjuvant LR6 experienced some advjuant assay interference as evidencedfrom the below detection level at both 4° C. and 25° C. However, theformulation adjuvanted with LR6 generated specific virus titer at 3months and is stable at 25° C.

TABLE 2 Viricidal and stability study of five adjuvants up to 3 months T= T = T = 1 T = 2 T = 4 T = 7 T = 3 Adjuvant 0 24 hr week week week weekmonths G1 - 4° C. 6.94 6.61 6.88 6.90 6.89 6.80 7.21 G1 - 25° C. 7.106.70 6.76 6.34 5.59 4.24 Below detection G2 - 4° C. 6.97 6.51 7.04 6.966.97 6.90 7.07 G2 - 25° C. 6.91 6.40 6.94 6.93 6.69 6.56 5.85 G3 - 4° C.6.68 6.25 6.74 6.80 6.74 6.93 6.92 G3 - 25° C. 6.42 6.40 6.71 6.69 6.676.63 5.85 G4 - 4° C. 6.51 Below 6.69 5.36 Below Below 6.78 Detection*Detection* Detection* G4 - 25° C. 6.52 Below 6.69 5.44 Below Below 6.21Detection* Detection* Detection* G5 - 4° C. 6.82 6.81 6.91 6.80 6.967.17 7.13 G5 - 25° C. 6.87 6.83 6.92 6.51 6.61 6.42 5.85 G6 - 4° C. 6.846.79 6.95 6.77 6.73 7.11 7.16 G6 - 25° C. 6.84 6.84 6.81 6.72 BelowBelow Below detection detection detection Below Detection*: adjuvantassay interference

The corresponding serology study was conducted in cattle. Each groupcontained 10 animals per experimental group (5 in control) and wasadministered one 2 ml dose at day zero followed by a boost at day 21.Blood samples were taken at several timepoints throughout the study andthe serum samples were analyzed. Serology by Virus Neutralizing Titerwas conducted. The initial data indicated serological responses at day14 post first vaccination in several groups. Taken together, the dataindicates that adenovirus-vectored FMD vaccines formulated in certainadjuvants may provide opportunities for thermostability improvements andability to handle temperature excursions. When combined with theseinitial data, the immunological response is either maintained orpotentially improved.

Example 4 Serology Immunogenicity of Two-Dose Vaccinatnion with MultipleRecombinant Human Adenovirus Vectored FMDV O1M Antigen in Cattle

The goal of the study is to assess the serological antibody response incattle following the administration of Adeno Vectored FMDV O1Manisaformulated with and without different adjuvants.

Fifty-five conventionally reared calves (approximately 5 months of age)were each randomized to one of six treatment groups as presented inTable 4 below.

TABLE 4 Route of Frequencey No. Adminis- of Adminis- of ani- GroupVaccine Adjuvant tration tration mals 1 Adt. ENABL ® IM Twice 21; 10O1/Manisa C1 days apart 2 Adt. LF IM Twice 21; 10 O1/Manisa days apart 3Adt. Polyacrylic IM Twice 21; 10 O1/Manisa acid days apart 4 Adt.Carbigen M IM Twice 21; 10 O1/Manisa days apart 5 Adt. None IM Twice 21;10 O1/Manisa days apart 6 FFB Placebo None IM Twice 21; 5 days apart

All calves except those from Group 6 (sentinels) were vaccinated with anAdenovirus vectored O1/Manisa construct with (Groups 1-4) or without anadjuvant (Groups 5), twice at a 21-day interval with 2 ml of the testvaccine. All injections were given via the intramuscular route (IM) overthe shoulder alternately on the right and left sides. Table 4 abovecontains a summary of the treatment for each group.

Calves were intermittently observed for at least 1 hour following eachvaccination for clinical signs of acute systemic adverse events. Bloodsamples were collected from all cattle on Days −1 (prior tovaccination), 7, 15, 21 (prior to vaccination), 28, and 35. Serumsamples from all cattle were tested for FMDV antibodies by Serum VirusNeutralization (SVN). In addition, antibody responses to Adenovirus(SAV) were determined in all animals from all groups in samplescollected on Day −1 and 35. The results for SVN and SAV were reported inLog₁₀ and a value≤0.6 Log₁₀ was considered negative for serum antibody.

Post-vaccination safety assessments included rectal temperature, visualinspection and palpation of injection sites for at least 3 daysfollowing each vaccination. Cows with local injection site adverseevents were observed intermittently until resolution of the abnormality.The study was terminated on D35 after the final blood collection.

Results of FMDV serological response using FMDV antibodies by SerumVirus Neutralization (SVN) Log₁₀ are described below.

All calves from all Groups tested negative for FMDV antibodies prior tothe start of the study. All sentinels were negative for FMDV antibodiesthroughout the study. Seroconversion following vaccination was definedas an increase Log₁₀ titer>0.9. By Day 14 (2 weeks following the 1stvaccination), 5/10 calves (50%) in the ENABL® C1 (Group 1) hadseroconverted. Those in the remaining vaccinated groups (2-5) hadbetween 20-40% of the calves seroconvert. In addition, the mean antibodytiter per group was slightly higher in group 1 (ENABL® C1) followed byGroups 2 (LF) and 3 (Polyacrylic acid) (FIG. 13).

By Day 35 (2 weeks following the 2nd vaccination), all vaccinatedanimals (Groups 1 [ENABL®], group 2 [LF] and group 5 [no adjuvant]) hadseroconverted followed by ninety and eighty percent of those in groups 3(Polyacrylic acid) and 4 (Carbigen M) respectively. Animals vaccinatedwith the LF, adjuvant, followed by those vaccinated without adjuvant(Group 5) and by those vaccinated with the ENABL® C1 (Group 1) adjuvanthad a higher antibody response following a two-dose vaccination regime(see FIG. 13).

Results of FMDV serological response using Adenovirus antibodies bySerum Virus Neutralization (SVN) Log₁₀ are described below.

All sentinels and vaccinated calves were negative to Adenovirus by SNantibody titers on Day −1 (≤0.6 Log₁₀). By day 35, all but one of thevaccinated calves (ID:134; Group 5) seroconverted with an overall highergeo mean titer by group in those vaccinated with a vaccine containingadjuvant (Groups 1-4) (see FIG. 14).

The results indicated serological responses at day 14 post firstvaccination in several groups. Two weeks after the second vaccinationthe higher antibody response was seen in vaccinated calves with the LFadjuvant followed by those vaccinated without adjuvant and with theEnable C1 adjuvant.

The results suggest that the antibody response following a singlevaccination regardless of the presence or absence of adjuvant is small.However, using a two vaccination (prime-boost) regimen the antibodyresponse is overall higher. There were no systemic adverse eventsobserved when the vaccine construct was administered twice (3 weeksapart) intramuscularly.

Example 5 Serology Assessment of FMDV Vaccines Following Vaccination inPigs

The goal of the study is to assess the antibody response in pigletsfollowing the administration of monovalent vaccine formulationscontaining Adeno 5 Vectored FMDV O1 Manisa) and/or FMDVbaculovirus-expressed O1 Manisa recombinant Virus Like Particle (VLP).

Twenty conventionally reared piglets (approximately 5 weeks of age) wererandomized to two treatment groups, each containing 10 piglets. Thegroup composition is presented in Table 5 below.

TABLE 5 dose per piglet; Adenovirus No. constructs of (FAID₅₀/2 ml Ani-Group Vaccines (log₁₀) Frequency mals 1 Adenovirus O1M 10⁸ Adenovirus 10No adjuvant/ X + 5 Bac O1M + O1M (Day 0) Adjuvant and Bac O1M TS6Adjuvant TS6 (Day 21) 2 Sentinel N/A N/A 10 (N/A)

Piglets in group 1 were vaccinated with 2 ml of the vaccine. Allinjections were given via the intramuscular route (IM) over the shoulderalternately on the right and left sides. Piglets were observed prior toeach vaccination for their overall health condition. Blood samples werecollected from all piglets on Days 0 (prior to vaccination), 7, 14, 21(prior to vaccination), 28 and 35. Day 35 serum samples from all pigletswere tested for FMDV antibodies by Serum Virus Neutralization (SVN).Samples from those piglets in group 1 were subject to SVN assay on allcollection days since they had an overall higher antibody responsefollowing on Day 35. The results were reported in Log₁₀ and a value 0.75Log₁₀ was considered negative for serum antibody. Post-vaccinationsafety assessments included rectal temperature, visual inspection andpalpation of injection sites for 3 days following each vaccination.

Results of FMDV serological response using FMDV antibodies by SerumVirus Neutralization (SVN) Log₁₀ are described below.

All sentinels were negative for FMDV antibodies prior to and at the endof the study. By Day 28 (1 week following the 2nd vaccination), allpiglets from Group 1 seroconverted (titers≥1.20 log₁₀) (see FIG. 15). Nosystemic and/or local adverse events attributable to vaccination wereobserved. The results clearly showed that even though vaccinated grouphad a small antibody response following the first vaccination, theantibody response following the second (prime-boost) vaccination washigh by the end of the study.

Example 6 Route of Administration

The goal of the study is to evaluate the serological response oftwo-dose vaccination in cattle or pigs when two recombinant Adenovirusvectored FMDV vaccines, one recombinant Adenovirus vectored FMDV vaccineand one baculovirus-expressed recombinant FMDV Virus like particle (VLP)vaccine, or two FMDV VLP vaccines are used, and when different routes ofadministration (transdermal, subcutaneous, or intradermal route ofadministration) are used. The study is also designed to addressinterference issue when multiple vaccines are administered.

The adjuvants are polyacrylic acid, LF2 emulsion, LR6 emulsion,CARBIGEN™ M and ENABL® C1. The treatment groups are represented in Table6 below.

TABLE 6 Administration Frequency of Group Vaccines route administration1 Adeno FMDV IM or IM/TD or Twice, 21 days IM/SQ apart 2 Adeno FMDV TDor TD/IM or Twice, 21 days TD/SQ apart 3 Adeno FMDV SQ or SQ/TD orTwice, 21 days SQ/IM apart 4 Baculo FMDV VLP IM or IM/TD or Twice, 21days IM/SQ apart 5 Baculo FMDV VLP TD or TD/IM or Twice, 21 days TD/SQapart 6 Baculo FMDV VLP SQ or SQ/TD or Twice, 21 days SQ/IM apart 7Adeno FMDV/Beculo IM or IM/TD or Twice, 21 days FMDV VLP IM/SQ apart 8Adeno FMDV/Beculo TD or TD/IM or Twice, 21 days FMDV VLP TD/SQ apart 9Adeno FMDV/Beculo SQ or SQ/TD or Twice, 21 days FMDV VLP SQ/IM apart

Calves are intermittently observed for at least 1 hour following eachvaccination for clinical signs of acute systemic adverse events. Bloodsamples are collected from all cattle on Days −1 (prior to vaccination),7, 15, 21 (prior to vaccination), 28, and 35. Serum samples from allcattle were tested for FMDV antibodies by Serum Virus Neutralization(SVN). In addition, antibody responses to Adenovirus (SAV) aredetermined in all animals from all groups in samples collected on Day −1and 35.

Post-vaccination safety assessments include rectal temperature, visualinspection and palpation of injection sites for at least 3 daysfollowing each vaccination. Cows with local injection site adverseevents are observed intermittently until resolution of the abnormality.

The results show a prime-boost affect for all groups but the prime-boosteffect is greater in animals that received a prime with the adenovirusvaccine followed by a boost with the baculovirus-FMD construct.Furthermore, the route of administration has an impact on serologicalresponse and protection, as well as duration of immunity. Specificroutes of administration and/or specific combination of administrationroutes TD, IM and SQ appear to exacerbate the immune response, furtherapplify protection, overcome interference, and protect MaternallyDerived Antibody-positive (MDA-positive) animals.

Example 7 Efficacy in Swine

Pigs are vaccinated against FMDV (against several setotypes) twice with21-days apart in a prime-boost regimen that looked at a heterologousprime-boost protocol (adeno prime followed by baculo boost) as well ashomologous prime boost (adeno-adeno; baculo-baculo), and challenged 14dpv by many FMDV serotypes, such as A24, A12, O1, Asia, Irn, and Iraqstrains.

In the dose titration study, the recombinant adeno-vectored FMDV vaccineis evaluated for the ability to confer protection against FMDgeneralized disease (pedal lesions) following homologous andheterologous challenges at 14 days post-vaccination (dpv). The proceduredescribed in Example 2 is used in this study to determine the minimumprotective dose in the prime-boost administration regimen.

The results demonstrate that the recombinant adenovector FMDV vaccineused in primer-boost protocol is highly immunogenic and efficaciousagainst homologous and heterologous FMDV challenges in pigs, overcomesinterference and protects Maternally Derived Antibody-positive(MDA-positive) animals.

Example 8 Efficacy in Bovine

Cattles are vaccinated first with a recombinant Adenovirus vectored FMDVvaccine and boosted with a conventional killed FMD vaccine or abaculovirus-expressed FMDV VLP vaccine 21-day apart and challenged atday 14 post second vaccination by many FMDV serotypes, such as A24, A12,O1, Asia, Irn, and Iraq strains.

In the dose titration study, the recombinant adeno-vectored FMDV vaccineis evaluated for the ability to confer protection against FMDgeneralized disease (pedal lesions) following direct homologous andheterologous challenges at 14 days post-vaccination (dpv). The proceduredescribed in Example 2 is used in this study to determine the minimumprotective dose in the prime-boost administration regimen.

The results demonstrate that the the prime-boost administration regimenis highly immunogenic and efficacious against homologous andheterologous FMDV challenges in cattle, and provides protection inanimals against FMDV infection, overcome interference, and protectsMaternally Derived Antibody-positive (MDA-positive) animals.

Having thus described in detail embodiments of the present disclosure,it is to be understood that the disclosure defined by the above examplesis not to be limited to particular details set forth in the abovedescription as many apparent variations thereof are possible withoutdeparting from the spirit or scope of the present disclosure.

All documents cited or referenced herein (“herein cited documents”), andall documents cited or referenced in herein cited documents, togetherwith any manufacturer's instructions, descriptions, productspecifications, and product sheets for any products mentioned herein orin any document incorporated by reference herein, are herebyincorporated herein by reference, and may be employed in the practice ofthe invention.

What we claim is:
 1. A composition or vaccine comprising a recombinantviral vector expressing a foot and mouth Disease Virus (FMDV) antigen,wherein the FMDV antigen comprises protein P1, protein 2A, protein 2B,protein 3B, and protein 3C, wherein the FMDV antigen comprises apolypeptide having at least 85% to 99% sequence identity to a sequenceselected from the group consisting of SEQ ID NOS: 4, 6, and 8, andwherein the composition or vaccine further comprises a pharmaceuticallyor veterinarily acceptable adjuvant.
 2. The composition or vaccine ofclaim 1, wherein the FMDV antigen comprises a polypeptide having atleast 90% to 99% sequence identity to a sequence selected from the groupconsisting of SEQ ID NOS: 4, 6, and
 8. 3. The composition or vaccine ofclaim 1, wherein the antigen further comprises at least a portion ofprotein 2C and protein 3A.
 4. The composition or vaccine of claim 1,wherein the antigen comprises a chimeric polypeptide comprising proteinP1 from a first FMDV strain and protein 3C from a second FMDV strain. 5.The composition or vaccine of claim 1, wherein the viral vector is anadenovirus.
 6. The composition or vaccine of claim 1, wherein thecomposition or vaccine further comprises a pharmaceutically orveterinarily acceptable carrier, vehicle or, excipient.
 7. Thecomposition or vaccine of claim 6, wherein composition or vaccine isstable out to 3 months when stored at 4° C.
 8. The composition orvaccine of claim 6, wherein the pharmaceutically or veterinarilyacceptable carrier, excipient, adjuvant, or vehicle is selected from thegroup consisting of polyacrylic acid, LF2 emulsion, LR6 emulsion, TS6emulsion, LR4 emulsion, carbomer, aluminum hydroxide, aluminumphosphate, saponin, CpG, water-in-oil emulsion, oil-in-water emulsion,carbomer-based adjuvant, and adjuvant composition comprising alipophile, a polymer of acrylic or methacrylic acid, saline,cholesterol, a saponin, and sodium hydroxide.
 9. A method of vaccinatingan animal susceptible to FMDV infection or eliciting an immune responsein the animal against FMDV comprising administering to the animal thevaccine according to claim
 1. 10. The method of claim 9, wherein themethod protects Maternally Derived Antibody-positive (MDA-positive)animals against FMDV infection.
 11. A method of vaccinating an animalsusceptible to FMDV infection or eliciting an immune response in theanimal against FMDV comprising: administering to the animal aprime-vaccine; and then administering to the animal a boost-vaccine,wherein at least one of the prime-vaccine and the boost-vaccine is thevaccine according to claim
 1. 12. The method of claim 11, wherein theprime-vaccine is the vaccine according to claim 1, wherein theboost-vaccine comprises an FMDV antigen, a recombinant viral vector thatexpresses, in vivo, an FMDV antigen, or both, and wherein the methodprotects the animal from FMDV and/or prevents FMDV disease progressionin the animal.
 13. The method of claim 11, wherein the prime-vaccinecomprises an FMDV antigen, a recombinant viral vector that expresses, invivo, and FMDV antigen, or both, wherein the boost-vaccine is thevaccine according to claim 1, and wherein the method protects the animalfrom FMDV and/or prevents FMDV disease progression in the animal.